Polynucleotides encoding engineered meganucleases having specificity for recognition sequences in the dystrophin gene

ABSTRACT

The present disclosure encompasses engineered meganucleases that bind and cleave recognition sequences within a dystrophin gene. The present disclosure also encompasses methods of using such engineered meganucleases to make genetically modified cells. Further, the disclosure encompasses pharmaceutical compositions comprising engineered meganuclease proteins, or polynucleotides encoding engineered meganucleases of the disclosure, and the use of such compositions for the modification of a dystrophin gene in a subject, or for treatment of Duchenne Muscular Dystrophy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of international application numberPCT/US2021/059146, filed Nov. 12, 2021, which claims the benefit of U.S.provisional application Nos. 63/113,131 and 63/233,664, filed Nov. 12,2020 and Aug. 16, 2021, respectively, each of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The application relates to the field of engineered meganucleases,molecular biology and recombinant nucleic acid technology. In particularaspects, the invention relates to engineered meganucleases useful forthe removal of exons from the dystrophin gene and for the treatment ofsubjects having Duchenne Muscular Dystrophy.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(P109070054US02-SEQ-EPG.xml; Size: 278,099 bytes; and Date of Creation:Sep. 13, 2022) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Duchenne Muscular Dystrophy (DMD) is a rare, X-linked muscledegenerative disorder that affects about 1 in every 3500 boys worldwide.The disease is caused by mutations in the dystrophin gene, which is thelargest known gene. The dystrophin gene spans 2.2 Mb of the X chromosomeand encodes predominantly a 14-kb transcript derived from 79 exons. Thefull-length dystrophin protein, as expressed in skeletal muscle, smoothmuscle, and cardiomyocytes, is 3685 amino acids and has a molecularweight of 427 kD. The severe Duchenne phenotype is generally associatedwith the loss of full-length dystrophin protein from skeletal andcardiac muscle, which leads to debilitating muscle degeneration and,ultimately, heart failure. A large number of different dystrophin genemutations have been described, many of them resulting in either thesevere DMD or the milder Becker Muscular Dystrophy.

There are several therapeutic strategies being pursued for the treatmentof DMD. First, “gene replacement” strategies are an active area ofresearch (Oshima et al. (2009) J. Am. Soc. Gene Ther. 17:73-80; Liu etal. (2005) Mol. Ther. 11:245-56; Lai et al. (2006) Hum Gene Ther.17:1036-42; Odom et al. (2008) Mol. Ther. 16:1539-45). This approachinvolves delivering a functional copy of the dystrophin gene to patientsusing a viral delivery vector, typically adeno-associated virus (AAV).The large size of the dystrophin gene makes it incompatible with thelimited carrying capacity of common viral vectors, however. Thisnecessitates the use of a “micro-dystrophin” gene in which most of therepetitive central portion of the gene is removed to leave only theminimally functional protein. It is not clear, however, that expressionof “micro-dystrophin” is sufficient for clinical benefit. In addition,this approach suffers from the possibility of random gene integrationinto the patient genome, which could lead to insertional mutagenesis,and the potential for immune reactions against the delivery vector.

A second approach to treating DMD involves the transplantation ofhealthy muscle precursor cells into patient muscle fibers (Peault et al.(2007) Mol. Ther. 15:867-77; Skuk et al. (2007) Neuromuscul. Disord.17:38-46). This approach suffers from inefficient migration of thetransplanted myoblasts and the potential for immune rejection by thepatient.

A third approach involves suppression of nonsense mutations using PTC124(Welch et al. (2007) Nature 447:87-91). This would require lifelongdosing of the drug, however, and the approach is yet to show anysignificant clinical benefit.

A fourth approach for treating DMD is called “Exon Skipping” (Williamset al. (2008) BMC Biotechnol. 8:35; Jearawiriyapaisarn et al. (2008) MolTher. 16:1624-29; Yokota et al. (2007) Acta Myol. 26:179-84; vanDeutekom et al. (2001) Hum. Mol. Gen. 10:1547-54; Benedetti et al.(2013) FEBS J. 280:4263-80; Rodino-Klapac (2013) Curr Neurol NeurosciRep. 13:332; Verhaart & Aartsma-Rus (2012) Curr Opin Neurol. 25:588-96).In general, the amino (N)- and carboxy (C)-terminal portions of thedystrophin gene are essential for its role as a “scaffold” protein thatmaintains membrane integrity in muscle fibres, whereas the central “roddomain”, which comprises 24 spectrin-like repeats, is at least partiallydispensable. Indeed, the severe Duchenne phenotype is typicallyassociated with mutations in the dystrophin gene that introduceframeshifts and/or premature termination codons, resulting in atruncated form of the dystrophin protein lacking the essentialC-terminal domain. Mutations in the central rod domain, including largedeletions of whole exons, typically result in the much milder Beckerphenotype if they maintain the reading frame such that the C-terminaldomain of the protein is intact.

DMD is most frequently caused by the deletion of one or more wholeexon(s), resulting in reading frame shift. For example, Exon 45 isfrequently deleted in Duchenne patients. Because Exon 45 is 176 bp long,which is not divisible by three, deleting the exon shifts Exons 46-79into the wrong reading frame. The same can be said of Exon 44, which is148 bp in length. However, if Exons 44 and 45 are deleted, the totalsize of the deletion is 324 bp, which is divisible by three. Thus, thedeletion of both exons does not result in a reading frame shift. Becausethese exons encode a portion of the non-essential rod domain of thedystrophin protein, deleting them from the protein is expected to resultin a mild Becker-like phenotype. Thus, a patient with the Duchennephenotype due to the deletion of one or more exon(s) can, potentially,be treated by eliminating one or more adjacent exons to restore thereading frame. This is the principle behind “Exon Skipping,” in whichmodified oligonucleotides are used to block splice acceptor sites indystrophin pre-mRNA so that one or more specific exons are absent fromthe processed transcript. The approach has been used to restoredystrophin gene expression in the mdx mouse model by skipping Exon 23,which harbored a disease-inducing nonsense mutation (Mann et al. (2001)Proc. Nat. Acad. Sci. USA 98:42-47). Oligonucleotide analogs that induceskipping of Exon 51 have also shown promise in early human clinicaltrials (Benedetti et al. (2013) FEBS J. 280:4263-80). The majorlimitations with this approach are: (1) the exon-skipping process isinefficient, resulting in relatively low levels of functional dystrophinexpression; and (2) the exon-skipping oligonucleotide has a relativelyshort half-life so the affect is transient, necessitating repeated andlife-long dosing. Thus, while Exon-Skipping approaches have shown somepromise in clinical trials, the improvements in disease progression havebeen minimal and variable.

The present disclosure improves upon current Exon-Skipping approaches bycorrecting gene expression at the level of the genomic DNA rather thanpre-mRNA. The invention is a permanent treatment for DMD that involvesthe excision of specific exons from the dystrophin coding sequence usinga pair of engineered, site-specific homing endonucleases, often referredto as meganucleases. By targeting a pair of such endonucleases to sitesin the intronic regions flanking exons in the dystrophin gene, it ispossible to permanently remove the intervening fragment from the genome.The resulting cell, and its progeny, will express a modified dystrophinin which a portion of the non-essential spectrin repeat domain isremoved but the essential N- and C-terminal domains are intact.

Homing endonucleases, or meganucleases, are a group ofnaturally-occurring nucleases that recognize 15-40 base-pair cleavagesites commonly found in the genomes of plants and fungi. They arefrequently associated with parasitic DNA elements, such as group 1self-splicing introns and inteins. They naturally promote homologousrecombination or gene insertion at specific locations in the host genomeby producing a double-stranded break in the chromosome, which recruitsthe cellular DNA-repair machinery (Stoddard (2006) Q. Rev. Biophys.38:49-95). Homing endonucleases are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cys box family and theHNH family. These families are characterized by structural motifs, whichaffect catalytic activity and recognition sequence. For instance,members of the LAGLIDADG family are characterized by having either oneor two copies of the conserved LAGLIDADG motif (see, Chevalier et al.(2001) Nucleic Acids Res. 29:3757-74). The LAGLIDADG homingendonucleases with a single copy of the LAGLIDADG motif form homodimers,whereas members with two copies of the LAGLIDADG motif are found asmonomers.

I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG family of homingendonucleases that recognizes and cuts a 22 basepair recognitionsequence in the chloroplast chromosome of the algae Chlamydomonasreinhardtii. Genetic selection techniques have been used to modify thewild-type I-CreI cleavage site preference (Sussman et al. (2004) J. Mol.Biol. 342:31-41; Chames et al. (2005) Nucleic Acids Res. 33:e178;Seligman et al. (2002) Nucleic Acids Res. 30:3870-79, Arnould et al.(2006) J. Mol. Biol. 355:443-58). Methods of rationally-designingmono-LAGLIDADG homing endonucleases have been described which arecapable of comprehensively redesigning 1-CreI and other homingendonucleases to target widely-divergent DNA sites, including sites inmammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineeredderivatives are normally dimeric but can be fused into a singlepolypeptide using a short peptide linker that joins the C-terminus of afirst subunit to the N-terminus of a second subunit (Li et al. (2009)Nucleic Acids Res. 37:1650-62; Grizot et al. (2009) Nucleic Acids Res.37:5405-19). Thus, a functional “single-chain” meganuclease can beexpressed from a single transcript. By delivering genes encoding twodifferent single-chain meganucleases to the same cell, it is possible tosimultaneously cut two different sites. This, coupled with the extremelylow frequency of off-target cutting observed with engineeredmeganucleases makes them the preferred endonuclease for the presentdisclosure.

SUMMARY OF THE INVENTION

The present disclosure provides engineered meganucleases that bind andcleave recognition sequences in a dystrophin gene (e.g., a humandystrophin gene), as well as compositions comprising such engineeredmeganucleases and methods of their use. In some embodiments, pairs ofengineered meganucleases are used to remove multiple exons from adystrophin gene by generating a first cleavage site in an intronupstream of a first exon and a second cleavage site in an introndownstream of a second exon. In particular examples described herein,the first cleavage site is generated in the intron 5′ upstream of exon45 of the dystrophin gene, while the second cleavage site is generatedin the intron 3′ downstream of exon 55. This process allows for excisionand removal of exons 45-55 from the dystrophin gene following annealmentof the two cleavage sites and repair of the genome. The recognitionsequences targeted by the disclosed engineered meganucleases areselected to have identical four basepair center sequences, such that thefirst and second cleavage sites will have complementary four basepair 3′overhangs that can perfectly ligate to one another (i.e., each basepairof one overhang pairs with its complement on the other overhang). Byremoving exons 45-55 from a mutant dystrophin gene that lacks one ormore of these exons, this approach results in a restoration of thenormal (i.e., wild-type) reading frame of the dystrophin gene. Cells sotreated will express a shortened modified form of the dystrophin proteinin which a portion of the central spectrin repeat domain is absent butthe N- and C-terminal domains are intact. This will, in many cases,reduce the severity of the disease. In some cases, it will result in amilder Becker phenotype.

Thus, in one aspect, the invention provides an engineered meganucleasethat binds and cleaves a recognition sequence in a dystrophin gene,wherein the engineered meganuclease comprises a first subunit and asecond subunit, wherein the first subunit binds to a first recognitionhalf-site of the recognition sequence and comprises a firsthypervariable (HVR1) region, and wherein the second subunit binds to asecond recognition half-site of the recognition sequence and comprises asecond hypervariable (HVR2) region.

In some embodiments, the recognition sequence comprises SEQ ID NO: 6.

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 24-79 of any one of SEQ ID NOs: 36-44. In someembodiments, the HVR1 region comprises one or more residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 36-44. In some embodiments,the HVR1 region comprises residues corresponding to residues 24, 26, 28,30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ IDNOs: 36-44. In some embodiments, the HVR1 region comprises Y, R, K. or Dat a residue corresponding to residue 66 of any one of SEQ ID NOs:36-44. In some embodiments, the HVR1 region comprises residues 24-79 ofany one of SEQ ID NOs: 36-44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 amino acid substitutions. In some embodiments, the HVR1 regioncomprises residues 24-79 of any one of SEQ ID NOs: 36-44.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 7-153 of any one ofSEQ ID NOs: 36-44. In some embodiments, the first subunit comprises G,S, or A at a residue corresponding to residue 19 of any one of SEQ IDNOs: 36-44. In some embodiments, the first subunit comprises a residuecorresponding to residue 19 of any one of SEQ ID NOs: 36-44. In someembodiments, the first subunit comprises E, Q, or K at a residuecorresponding to residue 80 of any one of SEQ TD NOs: 36-44. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of any one of SEQ ID NOs: 38, 39, or 149. In someembodiments, the first subunit comprises residues 7-153 of any one ofSEQ ID NOs: 36-44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acid substitutions. In some embodiments, the first subunitcomprises residues 7-153 of any one of SEQ ID NOs: 36-44.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 215-270 of any one of SEQ ID NOs: 36-44. Insome embodiments, the HVR2 region comprises one or more residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 36-44. Insome embodiments, the HVR2 region comprises residues corresponding toresidues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259,261, 266, and 268 of any one of SEQ ID NOs: 36-44. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of any one of SEQ ID NOs: 36-44. In some embodiments, theHVR2 region comprises a residue corresponding to residue 236 of SEQ IDNO: 39. In some embodiments, the HVR2 region comprises a residuecorresponding to residue 239 of SEQ ID NO: 37. In some embodiments, theHVR2 region comprises a residue corresponding to residue 241 of any oneof SEQ ID NOs: 36-37. In some embodiments, the HVR2 region comprises aresidue corresponding to residue 263 of SEQ ID NO: 36. In someembodiments, the HVR2 region comprises a residue corresponding toresidue 264 of any one of SEQ ID NOs: 36-44. In some embodiments, theHVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 36-44with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of any one of SEQ ID NOs: 36-44.

In some such embodiments, the second subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 198-344 of any oneof SEQ ID NOs: 36-44. In some embodiments, the second subunit comprisesG, S, or A at a residue corresponding to residue 210 of any one of SEQID NOs: 36-44. In some embodiments, the second subunit comprises E, Q,or K at a residue corresponding to residue 271 of any one of SEQ ID NOs:36-44. In some embodiments, the second subunit comprises a residuecorresponding to residue 271 of any one of SEQ ID NOs: 36, 39, 40, 43,or 44. In some embodiments, the second subunit comprises a residuecorresponding to residue 330 of any one of SEQ ID NOs: 36-38 or 40-44.In some embodiments, the second subunit comprises residues 198-344 ofany one of SEQ ID NOs: 36-44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 amino acid substitutions. In some embodiments, the secondsubunit comprises residues 198-344 of any one of SEQ ID NOs: 36-44.

In some such embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity any one of SEQ ID NOs: 36-44. In some embodiments, theengineered meganuclease comprises an amino acid sequence of any one ofSEQ ID NOs: 36-44. In some embodiments, the engineered meganuclease isencoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleicacid sequence set forth in any one of SEQ ID NOs: 60-68. In someembodiments, the engineered meganuclease is encoded by a nucleic acidsequence set forth in any one of SEQ ID NOs: 60-68.

In some embodiments, the recognition sequence comprises SEQ ID NO: 10.

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 24-79 of any one of SEQ ID NOs: 45-52. In someembodiments, the HVR1 region comprises one or more residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 45-52. In some embodiments,the HVR1 region comprises residues corresponding to residues 24, 26, 28,30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ IDNOs: 45-52. In some embodiments, the HVR1 region comprises Y, R, K, or Dat a residue corresponding to residue 66 of any one of SEQ ID NOs:45-52. In some embodiments, the HVR1 region comprises residues 24-79 ofany one of SEQ ID NOs: 45-52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11 amino acid substitutions. In some embodiments, the HVR1 regioncomprises residues 24-79 of any one of SEQ ID NOs: 45-52.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 7-153 of any one ofSEQ ID NOs: 45-52. In some embodiments, the first subunit comprises G,S, or A at a residue corresponding to residue 19 of any one of SEQ TDNOs: 45-52. In some embodiments, the first subunit comprises a residuecorresponding to residue 19 of any one of SEQ ID NOs: 45-52. In someembodiments, the first subunit comprises E, Q, or K at a residuecorresponding to residue 80 of any one of SEQ ID NOs: 45-52. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of any one of SEQ ID NOs: 45-51. In some embodiments, thefirst subunit comprises residues 7-153 of any one of SEQ ID NOs: 45-52with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of any one of SEQ ID NOs: 45-52.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 215-270 of any one of SEQ ID NOs: 45-52. Insome embodiments, the HVR2 region comprises one or more residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 45-52. Insome embodiments, the HVR2 region comprises residues corresponding toresidues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259,261, 266, and 268 of any one of SEQ ID NOs: 45-52. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of any one of SEQ ID NOs: 45-52. In some embodiments, theHVR2 region comprises residues corresponding to residues 239, 241, and264 of any one of SEQ ID NOs: 45-52. In some embodiments, the HVR2region comprises a residue corresponding to residue 250 of SEQ ID NO:45. In some embodiments, the HVR2 region comprises a residuecorresponding to residue 263 or any one of SEQ ID NOs: 45 or 46. In someembodiments, the HVR2 region comprises residues 215-270 of any one ofSEQ ID NOs: 45-52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 aminoacid substitutions. In some embodiments, the HVR2 region comprisesresidues 215-270 of any one of SEQ ID NOs: 45-52.

In some such embodiments, the second subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 198-344 of any oneof SEQ ID NOs: 45-52. In some embodiments, the second subunit comprisesG, S, or A at a residue corresponding to residue 210 of any one of SEQID NOs: 45-52. In some embodiments, the second subunit comprises E, Q,or K at a residue corresponding to residue 271 of any one of SEQ ID NOs:45-52. In some embodiments, the second subunit comprises a residuecorresponding to residue 271 of SEQ ID NO: 52. In some embodiments, thesecond subunit comprises a residue corresponding to residue 330 of anyone of SEQ ID NOs: 45-52. In some embodiments, the second subunitcomprises residues 198-344 of any one of SEQ ID NOs: 45-52 with up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In someembodiments, the second subunit comprises residues 198-344 of any one ofSEQ ID NOs: 45-52.

In some such embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity any one of SEQ ID NOs: 45-52. In some embodiments, theengineered meganuclease comprises an amino acid sequence of any one ofSEQ ID NOs: 45-52. In some embodiments, the engineered meganuclease isencoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleicacid sequence set forth in any one of SEQ ID NOs: 69-76. In someembodiments, the engineered meganuclease is encoded by a nucleic acidsequence set forth in any one of SEQ ID NOs: 69-76.

In some embodiments, the recognition sequence comprises SEQ ID NO: 12.

In some such embodiments, the HVR1 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 24-79 of any one of SEQ ID NOs: 53-59. In someembodiments, the HVR1 region comprises one or more residuescorresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46,68, 70, 75, and 77 of any one of SEQ ID NOs: 53-59. In some embodiments,the HVR1 region comprises residues corresponding to residues 24, 26, 28,30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ IDNOs: 53-59. In some embodiments, the HVR1 region comprises Y, R, K, or Dat a residue corresponding to residue 66 of any one of SEQ ID NOs:53-59. In some embodiments, the HVR1 region comprises a residuecorresponding to residue 64 of SEQ ID NO: 54. In some embodiments, theHVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 53-59with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of any one of SEQ ID NOs: 53-59.

In some such embodiments, the first subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 7-153 of any one ofSEQ ID NOs: 53-59. In some embodiments, the first subunit comprises G,S, or A at a residue corresponding to residue 19 of any one of SEQ IDNOs: 53-59. In some embodiments, the first subunit comprises a residuecorresponding to residue 19 of any one of SEQ ID NOs: 53-59. In someembodiments, the first subunit comprises E, Q, or K at a residuecorresponding to residue 80 of any one of SEQ ID NOs: 53-59. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of any one of SEQ ID NOs: 53-55, 57, or 58. In someembodiments, the first subunit comprises residues 7-153 of any one ofSEQ ID NOs: 53-59 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acid substitutions. In some embodiments, the first subunitcomprises residues 7-153 of any one of SEQ ID NOs: 53-59.

In some such embodiments, the HVR2 region comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to an amino acid sequencecorresponding to residues 215-270 of any one of SEQ ID NOs: 53-59. Insome embodiments, the HVR2 region comprises one or more residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 53-59. Insome embodiments, the HVR2 region comprises residues corresponding toresidues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259,261, 266, and 268 of any one of SEQ ID NOs: 53-59. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of any one of SEQ ID NOs: 53-59. In some embodiments, theHVR2 region comprises a residue corresponding to residue 239 of SEQ IDNO: 53 or SEQ ID NO: 55. In some embodiments, the HVR2 region comprisesa residue corresponding to residue 241 of any one of SEQ ID NOs: 53-55.In some embodiments, the HVR2 region comprises a residue correspondingto residue 255 of SEQ ID NO: 55. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of any one of SEQ IDNOs: 56-59. In some embodiments, the HVR2 region comprises a residuecorresponding to residue 264 of any one of SEQ ID NOs: 53-59. In someembodiments, the HVR2 region comprises residues 215-270 of any one ofSEQ ID NOs: 53-59 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 aminoacid substitutions. In some embodiments, the HVR2 region comprisesresidues 215-270 of any one of SEQ ID NOs: 53-59.

In some such embodiments, the second subunit comprises an amino acidsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to residues 198-344 of any oneof SEQ ID NOs: 53-59. In some embodiments, the second subunit comprisesG, S, or A at a residue corresponding to residue 210 of any one of SEQID NOs: 53-59. In some embodiments, the second subunit comprises E, Q,or K at a residue corresponding to residue 271 of any one of SEQ ID NOs:53-59. In some embodiments, the second subunit comprises a residuecorresponding to residue 271 of any one of SEQ ID NOs: 53 or 55-59. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of any one of SEQ ID NOs: 54-59. In some embodiments, thesecond subunit comprises residues 198-344 of any one of SEQ ID NOs:53-59 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of any one of SEQ ID NOs: 53-59.

In some such embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity any one of SEQ ID NOs: 53-59. In some embodiments, theengineered meganuclease comprises the amino acid sequence of any one ofSEQ ID NOs: 53-59. In some embodiments, the engineered meganuclease isencoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleicacid sequence set forth in any one of SEQ ID NOs: 77-83. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence set forth in any one of SEQ ID NOs: 77-83.

In each of the embodiments above, the engineered meganuclease cancomprise a nuclear localization signal. In some embodiments, the nuclearlocalization signal is at the N-terminus of the engineered meganuclease.In some embodiments, the nuclear localization signal comprises an aminoacid sequence having at least 80% or at least 90% sequence identity toSEQ ID NO: 3. In some embodiments, the nuclear localization signalcomprises SEQ ID NO: 3.

In another aspect, the invention provides a polynucleotide comprising anucleic acid sequence encoding an engineered meganuclease describedherein. In some embodiments, the polynucleotide is an mRNA.

In another aspect, the invention provides a recombinant DNA constructcomprising a polynucleotide comprising a nucleic acid sequence encodingan engineered meganuclease described herein.

In some embodiments, the recombinant DNA construct encodes a recombinantvirus comprising the polynucleotide. In some embodiments, therecombinant virus is a recombinant adenovirus, a recombinant lentivirus,a recombinant retrovirus, or a recombinant AAV. In some embodiments, therecombinant virus is a recombinant AAV. In some embodiments, therecombinant AAV has an rh.74 capsid. In some embodiments, therecombinant AAV has an AAV9 capsid. In some embodiments, the rh.74capsid comprises an amino acid sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ IDNO: 182. In some embodiments, the rh.74 capsid comprises an amino acidsequence of SEQ ID NO: 182. In some embodiments, the AAV9 capsidcomprises an amino acid sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:183. In some embodiments, the AAV9 capsid comprises an amino acidsequence of SEQ ID NO: 183. In some embodiments, the recombinant AAV hasan AAV8 capsid.

In some embodiments, the nucleic acid sequence comprises a promoteroperably linked to the nucleic acid sequence encoding an engineeredmeganuclease described herein. In some embodiments, the promoter is amuscle-specific promoter. In some embodiments, the muscle-specificpromoter comprises an MCK promoter, a C5-12 promoter, a spc 5-12promoter, a MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, a SP-301promoter, a SP-817 promoter, or a SP-905 promoter. In some embodiments,the promoter is capable of expressing an engineered meganucleasedescribed herein in a muscle precursor cell (e.g., a satellite cell orstem cell).

In another aspect, the invention provides a recombinant virus comprisinga polynucleotide comprising a nucleic acid sequence encoding anengineered meganuclease described herein.

In some embodiments, the recombinant virus is a recombinant adenovirus,a recombinant lentivirus, a recombinant retrovirus, or a recombinantAAV. In some embodiments, the recombinant virus is a recombinant AAV. Insome embodiments, the recombinant AAV has an rh.74 capsid. In someembodiments, the rh.74 capsid comprises an amino acid sequence having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to SEQ ID NO: 182. In some embodiments, the rh.74 capsidcomprises an amino acid sequence of SEQ ID NO: 182. In some embodiments,the recombinant AAV has an AAV9 capsid. In some embodiments, the AAV9capsid comprises an amino acid sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ IDNO: 183. In some embodiments, the AAV9 capsid comprises an amino acidsequence of SEQ ID NO: 183. In some embodiments, the recombinant AAV hasan AAV8 capsid.

In some embodiments, the polynucleotide comprises a promoter operablylinked to the nucleic acid sequence encoding an engineered meganucleasedescribed herein. In some embodiments, the promoter is a muscle-specificpromoter. In some embodiments, the muscle-specific promoter comprises anMCK promoter, a C5-12 promoter, a spc 5-12 promoter, a MHCK7 promoter, aCK8 promoter, a SK-CRM4 promoter, a SP-301 promoter, a SP-817 promoter,or a SP-905 promoter. In some embodiments, the promoter is capable ofexpressing an engineered meganuclease described herein in a muscleprecursor cell (e.g., a satellite cell or stem cell).

In another aspect, the invention provides a lipid nanoparticlecomposition comprising lipid nanoparticles comprising a polynucleotide,wherein the polynucleotide comprises a nucleic acid sequence encoding anengineered meganuclease described herein. In some embodiments, thepolynucleotide is an mRNA.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an engineeredmeganuclease described herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a polynucleotidedescribed herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a recombinant DNAconstruct described herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a recombinant virusdescribed herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a lipidnanoparticle composition described herein.

In another aspect, the invention provides a polynucleotide comprising afirst nucleic acid sequence encoding a first engineered meganuclease anda second nucleic acid sequence encoding a second engineeredmeganuclease, wherein the first engineered meganuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and wherein the second engineeredmeganuclease is an engineered meganuclease described herein that bindsand cleaves a recognition sequence comprising SEQ ID NO: 10, or whereinthe second engineered meganuclease is an engineered meganucleasedescribed herein that binds and cleaves a recognition sequencecomprising SEQ ID NO: 12.

In some embodiments, the first engineered meganuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered meganucleaseis an engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 10. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease areselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 1.

TABLE 1 Recognition Sequence of Recognition Sequence of SEQ ID NO: 6 SEQID NO: 10 Combination First Meganuclease SEQ ID NO: Second MeganucleaseSEQ ID NO: 1 DMD 19-20x.13 36 DMD 35-36x.63 45 2 DMD 19-20x.87 37 DMD35-36x.63 45 3 DMD 19-20L.249 38 DMD 35-36x.63 45 4 DMD 19-20L.302 39DMD 35-36x.63 45 5 DMD 19-20L.329 40 DMD 35-36x.63 45 6 DMD 19-20L.37441 DMD 35-36x.63 45 7 DMD 19-20L.375 42 DMD 35-36x.63 45 8 DMD19-20L.431 43 DMD 35-36x.63 45 9 DMD 19-20L.458 44 DMD 35-36x.63 45 10DMD 19-20x.13 36 DMD 35-36x.81 46 11 DMD 19-20x.87 37 DMD 35-36x.81 4612 DMD 19-20L.249 38 DMD 35-36x.81 46 13 DMD 19-20L.302 39 DMD 35-36x.8146 14 DMD 19-20L.329 40 DMD 35-36x.81 46 15 DMD 19-20L.374 41 DMD35-36x.81 46 16 DMD 19-20L.375 42 DMD 35-36x.81 46 17 DMD 19-20L.431 43DMD 35-36x.81 46 18 DMD 19-20L.458 44 DMD 35-36x.81 46 19 DMD 19-20x.1336 DMD 35-36L.195 47 20 DMD 19-20x.87 37 DMD 35-36L.195 47 21 DMD19-20L.249 38 DMD 35-36L.195 47 22 DMD 19-20L.302 39 DMD 35-36L.195 4723 DMD 19-20L.329 40 DMD 35-36L.195 47 24 DMD 19-20L.374 41 DMD35-36L.195 47 25 DMD 19-20L.375 42 DMD 35-36L.195 47 26 DMD 19-20L.43143 DMD 35-36L.195 47 27 DMD 19-20L.458 44 DMD 35-36L.195 47 28 DMD19-20x.13 36 DMD 35-36L.282 48 29 DMD 19-20x.87 37 DMD 35-36L.282 48 30DMD 19-20L.249 38 DMD 35-36L.282 48 31 DMD 19-20L.302 39 DMD 35-36L.28248 32 DMD 19-20L.329 40 DMD 35-36L.282 48 33 DMD 19-20L.374 41 DMD35-36L.282 48 34 DMD 19-20L.375 42 DMD 35-36L.282 48 35 DMD 19-20L.43143 DMD 35-36L.282 48 36 DMD 19-20L.458 44 DMD 35-36L.282 48 37 DMD19-20x.13 36 DMD 35-36L.349 49 38 DMD 19-20x.87 37 DMD 35-36L.349 49 39DMD 19-20L.249 38 DMD 35-36L.349 49 40 DMD 19-20L.302 39 DMD 35-36L.34949 41 DMD 19-20L.329 40 DMD 35-36L.349 49 42 DMD 19-20L.374 41 DMD35-36L.349 49 43 DMD 19-20L.375 42 DMD 35-36L.349 49 44 DMD 19-20L.43143 DMD 35-36L.349 49 45 DMD 19-20L.458 44 DMD 35-36L.349 49 46 DMD19-20x.13 36 DMD 35-36L.376 50 47 DMD 19-20x.87 37 DMD 35-36L.376 50 48DMD 19-20L.249 38 DMD 35-36L.376 50 49 DMD 19-20L.302 39 DMD 35-36L.37650 50 DMD 19-20L.329 40 DMD 35-36L.376 50 51 DMD 19-20L.374 41 DMD35-36L.376 50 52 DMD 19-20L.375 42 DMD 35-36L.376 50 53 DMD 19-20L.43143 DMD 35-36L.376 50 54 DMD 19-20L.458 44 DMD 35-36L.376 50 55 DMD19-20x.13 36 DMD 35-36L.457 51 56 DMD 19-20x.87 37 DMD 35-36L.457 51 57DMD 19-20L.249 38 DMD 35-36L.457 51 58 DMD 19-20L.302 39 DMD 35-36L.45751 59 DMD 19-20L.329 40 DMD 35-36L.457 51 60 DMD 19-20L.374 41 DMD35-36L.457 51 61 DMD 19-20L.375 42 DMD 35-36L.457 51 62 DMD 19-20L.43143 DMD 35-36L.457 51 63 DMD 19-20L.458 44 DMD 35-36L.457 51 64 DMD19-20x.13 36 DMD 35-36L.469 52 65 DMD 19-20x.87 37 DMD 35-36L.469 52 66DMD 19-20L.249 38 DMD 35-36L.469 52 67 DMD 19-20L.302 39 DMD 35-36L.46952 68 DMD 19-20L.329 40 DMD 35-36L.469 52 69 DMD 19-20L.374 41 DMD35-36L.469 52 70 DMD 19-20L.375 42 DMD 35-36L.469 52 71 DMD 19-20L.43143 DMD 35-36L.469 52 72 DMD 19-20L.458 44 DMD 35-36L.469 52

In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.81 (SEQ ID NO: 46), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.63 (SEQ ID NO: 45), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.195 (SEQ ID NO: 47), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.282 (SEQ ID NO: 48), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.349 (SEQ ID NO: 49), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49), or a variantthereof described herein.

In certain embodiments, the first engineered meganuclease is anengineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 6, and the second engineeredmeganuclease is an engineered meganuclease described herein that bindsand cleaves a recognition sequence comprising SEQ ID NO: 12. In someembodiments, the first engineered meganuclease and the second engineeredmeganuclease are selected from the combinations of meganucleases (andvariants thereof described herein) provided in Table 2.

TABLE 2 Recognition Sequence of Recognition Sequence of SEQ ID NO: 6 SEQID NO: 12 Combination First Meganuclease SEQ ID NO: Second MeganucleaseSEQ ID NO: 1 DMD 19-20x.13 36 DMD 37-38x.15 46 2 DMD 19-20x.87 37 DMD37-38x.15 46 3 DMD 19-20L.249 38 DMD 37-38x.15 46 4 DMD 19-20L.302 39DMD 37-38x.15 46 5 DMD 19-20L.329 40 DMD 37-38x.15 46 6 DMD 19-20L.37441 DMD 37-38x.15 46 7 DMD 19-20L.375 42 DMD 37-38x.15 46 8 DMD19-20L.431 43 DMD 37-38x.15 46 9 DMD 19-20L.458 44 DMD 37-38x.15 46 10DMD 19-20x.13 36 DMD 37-38x.66 47 11 DMD 19-20x.87 37 DMD 37-38x.66 4712 DMD 19-20L.249 38 DMD 37-38x.66 47 13 DMD 19-20L.302 39 DMD 37-38x.6647 14 DMD 19-20L.329 40 DMD 37-38x.66 47 15 DMD 19-20L.374 41 DMD37-38x.66 47 16 DMD 19-20L.375 42 DMD 37-38x.66 47 17 DMD 19-20L.431 43DMD 37-38x.66 47 18 DMD 19-20L.458 44 DMD 37-38x.66 47 19 DMD 19-20x.1336 DMD 37-38x.79 48 20 DMD 19-20x.87 37 DMD 37-38x.79 48 21 DMD19-20L.249 38 DMD 37-38x.79 48 22 DMD 19-20L.302 39 DMD 37-38x.79 48 23DMD 19-20L.329 40 DMD 37-38x.79 48 24 DMD 19-20L.374 41 DMD 37-38x.79 4825 DMD 19-20L.375 42 DMD 37-38x.79 48 26 DMD 19-20L.431 43 DMD 37-38x.7948 27 DMD 19-20L.458 44 DMD 37-38x.79 48 28 DMD 19-20x.13 36 DMD37-38L.166 49 29 DMD 19-20x.87 37 DMD 37-38L.166 49 30 DMD 19-20L.249 38DMD 37-38L.166 49 31 DMD 19-20L.302 39 DMD 37-38L.166 49 32 DMD19-20L.329 40 DMD 37-38L.166 49 33 DMD 19-20L.374 41 DMD 37-38L.166 4934 DMD 19-20L.375 42 DMD 37-38L.166 49 35 DMD 19-20L.431 43 DMD37-38L.166 49 36 DMD 19-20L.458 44 DMD 37-38L.166 49 37 DMD 19-20x.13 36DMD 37-38L.478 57 38 DMD 19-20x.87 37 DMD 37-38L.478 57 39 DMD19-20L.249 38 DMD 37-38L.478 57 40 DMD 19-20L.302 39 DMD 37-38L.478 5741 DMD 19-20L.329 40 DMD 37-38L.478 57 42 DMD 19-20L.374 41 DMD37-38L.478 57 43 DMD 19-20L.375 42 DMD 37-38L.478 57 44 DMD 19-20L.43143 DMD 37-38L.478 57 45 DMD 19-20L.458 44 DMD 37-38L.478 57 46 DMD19-20x.13 36 DMD 37-38L.512 58 47 DMD 19-20x.87 37 DMD 37-38L.512 58 48DMD 19-20L.249 38 DMD 37-38L.512 58 49 DMD 19-20L.302 39 DMD 37-38L.51258 50 DMD 19-20L.329 40 DMD 37-38L.512 58 51 DMD 19-20L.374 41 DMD37-38L.512 58 52 DMD 19-20L.375 42 DMD 37-38L.512 58 53 DMD 19-20L.43143 DMD 37-38L.512 58 54 DMD 19-20L.458 44 DMD 37-38L.512 58 55 DMD19-20x.13 36 DMD 37-38L.528 59 56 DMD 19-20x.87 37 DMD 37-38L.528 59 57DMD 19-20L.249 38 DMD 37-38L.528 59 58 DMD 19-20L.302 39 DMD 37-38L.52859 59 DMD 19-20L.329 40 DMD 37-38L.528 59 60 DMD 19-20L.374 41 DMD37-38L.528 59 61 DMD 19-20L.375 42 DMD 37-38L.528 59 62 DMD 19-20L.43143 DMD 37-38L.528 59 63 DMD 19-20L.458 44 DMD 37-38L.528 59

In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.15 (SEQ ID NO: 53), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.66 (SEQ ID NO: 54), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.79 (SEQ ID NO: 55), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variantthereof described herein.

In some embodiments, the polynucleotide is an mRNA. In some embodiments,the first nucleic acid sequence and the second nucleic acid sequence areseparated by an IRES or 2A sequence. In certain embodiments, the 2Asequence is a T2A, P2A, E2A, or F2A sequence.

In another aspect, the invention provides a recombinant DNA constructcomprising a polynucleotide described herein (i.e., that comprises afirst nucleic acid sequence encoding a first engineered meganuclease anda second nucleic acid sequence encoding a second engineeredmeganuclease).

In some embodiments, the first nucleic acid sequence and the secondnucleic acid sequence are separated by an IRES or 2A sequence. Incertain embodiments, the 2A sequence is a T2A, P2A, E2A, or F2Asequence.

In some embodiments, the polynucleotide comprises a promoter operablylinked to the first nucleic acid sequence and the second nucleic acidsequence. In some embodiments, the promoter is a muscle-specificpromoter. In some embodiments, the muscle-specific promoter comprises anMCK promoter, a C5-12 promoter, a spc 5-12 promoter, a MHCK7 promoter, aCK8 promoter, a SK-CRM4 promoter, a SP-301 promoter, a SP-817 promoter,or a SP-905 promoter. In some embodiments, the promoter is capable ofexpressing a first and second engineered meganuclease described hereinin a muscle precursor cell (e.g., a satellite cell or stem cell).

In some embodiments, the polynucleotide comprises a first promoteroperably linked to the first nucleic acid sequence and a second promoteroperably linked to the second nucleic acid sequence. In someembodiments, the first promoter and the second promoter aremuscle-specific promoters. In some embodiments, the muscle-specificpromoters comprise an MCK promoter, a C5-12 promoter, a spc 5-12promoter, a MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, a SP-301promoter, a SP-817 promoter, a SP-905 promoter, or a combinationthereof. In some embodiments, the promoters are capable of expressing afirst and second engineered meganuclease described herein in a muscleprecursor cell (e.g., a satellite cell or stem cell).

In some embodiments, the recombinant DNA construct encodes a recombinantvirus comprising the polynucleotide. In some embodiments, therecombinant virus is a recombinant adenovirus, a recombinant lentivirus,a recombinant retrovirus, or a recombinant AAV. In some embodiments, therecombinant virus is a recombinant AAV. In some embodiments, therecombinant AAV has an rh.74 capsid. In some embodiments, therecombinant AAV has an AAV9 capsid. In some embodiments, the rh.74capsid comprises an amino acid sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ IDNO: 182. In some embodiments, the rh.74 capsid comprises an amino acidsequence of SEQ ID NO: 182. In some embodiments, the AAV9 capsidcomprises an amino acid sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:183. In some embodiments, the AAV9 capsid comprises an amino acidsequence of SEQ ID NO: 183. In some embodiments, the recombinant AAV hasan AAV8 capsid.

In another aspect, the invention provides a recombinant virus comprisinga polynucleotide described herein (i.e., that comprises a first nucleicacid sequence encoding a first engineered meganuclease and a secondnucleic acid sequence encoding a second engineered meganuclease).

In some embodiments, the polynucleotide comprises a promoter operablylinked to the first nucleic acid sequence and the second nucleic acidsequence. In some embodiments, the first nucleic acid sequence and thesecond nucleic acid sequence are separated by an IRES or 2A sequence. Incertain embodiments, the 2A sequence is a T2A, P2A, E2A, or F2Asequence.

In some embodiments, the promoter is a muscle-specific promoter. In someembodiments, the muscle-specific promoter comprises an MCK promoter, aC5-12 promoter, a spc 5-12 promoter, a MHCK7 promoter, a CK8 promoter, aSK-CRM4 promoter, a SP-301 promoter, a SP-817 promoter, or a SP-905promoter. In some embodiments, the promoter is capable of expressing anengineered meganuclease described herein in a muscle precursor cell(e.g., a satellite cell or stem cell).

In some embodiments, the polynucleotide comprises a first promoteroperably linked to the first nucleic acid sequence and a second promoteroperably linked to the second nucleic acid sequence. In someembodiments, the first promoter and the second promoter aremuscle-specific promoters. In some embodiments, the muscle-specificpromoters comprise an MCK promoter, a C5-12 promoter, a spc 5-12promoter, a MHCK7 promoter, a CK8 promoter, a SK-CRM4 promoter, a SP-301promoter, a SP-817 promoter, a SP-905 promoter, or a combinationthereof. In some embodiments, the promoters are capable of expressing anengineered meganuclease described herein in a muscle precursor cell(e.g., a satellite cell or stem cell). In some embodiments, therecombinant virus is a recombinant adenovirus, a recombinant lentivirus,a recombinant retrovirus, or a recombinant AAV. In some embodiments, therecombinant virus is a recombinant AAV. In some embodiments, therecombinant AAV has an rh.74 capsid. In some embodiments, therecombinant AAV has an AAV9 capsid. In some embodiments, the rh.74capsid comprises an amino acid sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ IDNO: 182. In some embodiments, the rh.74 capsid comprises an amino acidsequence of SEQ ID NO: 182. In some embodiments, the AAV9 capsidcomprises an amino acid sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:183. In some embodiments, the AAV9 capsid comprises an amino acidsequence of SEQ ID NO: 183. In some embodiments, the recombinant AAV hasan AAV8 capsid.

In another aspect, the invention provides a lipid nanoparticlecomposition comprising lipid nanoparticles comprising a polynucleotidedescribed herein (i.e., that comprises a first nucleic acid sequenceencoding a first engineered meganuclease and a second nucleic acidsequence encoding a second engineered meganuclease).

In some embodiments, the polynucleotide is an mRNA described herein. Insome embodiments, the polynucleotide is a recombinant DNA constructdescribed herein.

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a polynucleotidedescribed herein (i.e., that comprises a first nucleic acid sequenceencoding a first engineered meganuclease and a second nucleic acidsequence encoding a second engineered meganuclease).

In some embodiments, the polynucleotide comprises an mRNA describedherein. In some embodiments, the polynucleotide comprises a recombinantDNA construct described herein. In some embodiments, the pharmaceuticalcomposition comprises a recombinant virus described herein. In someembodiments, the pharmaceutical composition comprises a lipidnanoparticle composition described herein.

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell a polynucleotidecomprising a nucleic acid sequence encoding an engineered meganucleasedescribed herein, wherein the engineered meganuclease is expressed inthe eukaryotic cell, and wherein the engineered meganuclease produces acleavage site in the dystrophin gene at a recognition sequencecomprising SEQ ID NO: 6. In some embodiments, the eukaryotic cell is amammalian cell. In some embodiments, the mammalian cell is a musclecell. In some embodiments, the muscle cell is a muscle precursor cell(e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell. In some embodiments, the polynucleotide is introduced into theeukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus(e.g., a recombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell an engineeredmeganuclease described herein, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 6. In some embodiments, the eukaryoticcell is a mammalian cell. In some embodiments, the mammalian cell is amuscle cell. In some embodiments, the muscle cell is a muscle precursorcell (e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell.

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell one or more polynucleotides comprising: a first nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe engineered meganuclease is expressed in the eukaryotic cell; and asecond nucleic acid sequence comprising the sequence of interest,wherein the engineered meganuclease produces a cleavage site in thedystrophin gene at a recognition sequence comprising SEQ ID NO: 6, andwherein the sequence of interest is inserted into the dystrophin gene atthe cleavage site. In some embodiments, the second nucleic acid sequencecomprises nucleic acid sequences homologous to nucleic acid sequencesflanking the cleavage site and the sequence of interest is inserted atthe cleavage site by homologous recombination. In some embodiments, theeukaryotic cell is a mammalian cell. In some embodiments, the mammaliancell is a muscle cell. In some embodiments, the muscle cell is a muscleprecursor cell (e.g., a satellite cell or stem cell), a skeletal musclecell or a cardiac muscle cell. In some embodiments, the mammalian cellis a human cell. In some embodiments, the one or more polynucleotidesare introduced into the eukaryotic cell by lipid nanoparticles, mRNA, orrecombinant viruses (e.g., recombinant AAVs).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell an engineered meganuclease described herein, and a polynucleotidecomprising the sequence of interest, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 6, and wherein the sequence of interestis inserted into the dystrophin gene at the cleavage site. In someembodiments, the polynucleotide comprises nucleic acid sequenceshomologous to nucleic acid sequences flanking the cleavage site and thesequence of interest is inserted at the cleavage site by homologousrecombination. In some embodiments, the eukaryotic cell is a mammaliancell. In some embodiments, the mammalian cell is a muscle cell. In someembodiments, the muscle cell is a muscle precursor cell (e.g., asatellite cell or stem cell), a skeletal muscle cell or a cardiac musclecell. In some embodiments, the mammalian cell is a human cell. In someembodiments, the polynucleotide is introduced into the eukaryotic cellby a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., arecombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell a polynucleotidecomprising a nucleic acid sequence encoding an engineered meganucleasedescribed herein, wherein the engineered meganuclease is expressed inthe eukaryotic cell, and wherein the engineered meganuclease produces acleavage site in the dystrophin gene at a recognition sequencecomprising SEQ ID NO: 10. In some embodiments, the eukaryotic cell is amammalian cell. In some embodiments, the mammalian cell is a musclecell. In some embodiments, the muscle cell is a muscle precursor cell(e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell. In some embodiments, the polynucleotide is introduced into theeukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus(e.g., a recombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell an engineeredmeganuclease described herein, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 10. In some embodiments, the eukaryoticcell is a mammalian cell. In some embodiments, the mammalian cell is amuscle cell. In some embodiments, the muscle cell is a muscle precursorcell (e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell.

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell one or more polynucleotides comprising: a first nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe engineered meganuclease is expressed in the eukaryotic cell; and asecond nucleic acid sequence comprising the sequence of interest,wherein the engineered meganuclease produces a cleavage site in thedystrophin gene at a recognition sequence comprising SEQ TD NO: 10, andwherein the sequence of interest is inserted into the dystrophin gene atthe cleavage site. In some embodiments, the second nucleic acid sequencecomprises nucleic acid sequences homologous to nucleic acid sequencesflanking the cleavage site and the sequence of interest is inserted atthe cleavage site by homologous recombination. In some embodiments, theeukaryotic cell is a mammalian cell. In some embodiments, the mammaliancell is a muscle cell. In some embodiments, the muscle cell is a muscleprecursor cell (e.g., a satellite cell or stem cell), a skeletal musclecell or a cardiac muscle cell. In some embodiments, the mammalian cellis a human cell. In some embodiments, the one or more polynucleotidesare introduced into the eukaryotic cell by lipid nanoparticles, mRNA, orrecombinant viruses (e.g., recombinant AAVs).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell an engineered meganuclease described herein, and a polynucleotidecomprising the sequence of interest, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 10, and wherein the sequence of interestis inserted into the dystrophin gene at the cleavage site. In someembodiments, the polynucleotide comprises nucleic acid sequenceshomologous to nucleic acid sequences flanking the cleavage site and thesequence of interest is inserted at the cleavage site by homologousrecombination. In some embodiments, the eukaryotic cell is a mammaliancell. In some embodiments, the mammalian cell is a muscle cell. In someembodiments, the muscle cell is a muscle precursor cell (e.g., asatellite cell or stem cell), a skeletal muscle cell or a cardiac musclecell. In some embodiments, the mammalian cell is a human cell. In someembodiments, the polynucleotide is introduced into the eukaryotic cellby a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., arecombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell a polynucleotidecomprising a nucleic acid sequence encoding an engineered meganucleasedescribed herein, wherein the engineered meganuclease is expressed inthe eukaryotic cell, and wherein the engineered meganuclease produces acleavage site in the dystrophin gene at a recognition sequencecomprising SEQ ID NO: 12. In some embodiments, the eukaryotic cell is amammalian cell. In some embodiments, the mammalian cell is a musclecell. In some embodiments, the muscle cell is a muscle precursor cell(e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell. In some embodiments, the polynucleotide is introduced into theeukaryotic cell by a lipid nanoparticle, an mRNA, or a recombinant virus(e.g., a recombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell having a modified target sequencein a dystrophin gene of the genetically modified eukaryotic cell, themethod comprising: introducing into a eukaryotic cell an engineeredmeganuclease described herein, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 12. In some embodiments, the eukaryoticcell is a mammalian cell. In some embodiments, the mammalian cell is amuscle cell. In some embodiments, the muscle cell is a muscle precursorcell (e.g., a satellite cell or stem cell), a skeletal muscle cell or acardiac muscle cell. In some embodiments, the mammalian cell is a humancell.

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell one or more polynucleotides comprising: a first nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe engineered meganuclease is expressed in the eukaryotic cell; and asecond nucleic acid sequence comprising the sequence of interest,wherein the engineered meganuclease produces a cleavage site in thedystrophin gene at a recognition sequence comprising SEQ ID NO: 12, andwherein the sequence of interest is inserted into the dystrophin gene atthe cleavage site. In some embodiments, the second nucleic acid sequencecomprises nucleic acid sequences homologous to nucleic acid sequencesflanking the cleavage site and the sequence of interest is inserted atthe cleavage site by homologous recombination. In some embodiments, theeukaryotic cell is a mammalian cell. In some embodiments, the mammaliancell is a muscle cell. In some embodiments, the muscle cell is a muscleprecursor cell (e.g., a satellite cell or stem cell), a skeletal musclecell or a cardiac muscle cell. In some embodiments, the mammalian cellis a human cell. In some embodiments, the one or more polynucleotidesare introduced into the eukaryotic cell by lipid nanoparticles, mRNA, orrecombinant viruses (e.g., recombinant AAVs).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising an exogenous sequence ofinterest inserted into a dystrophin gene of the genetically modifiedeukaryotic cell, the method comprising introducing into a eukaryoticcell an engineered meganuclease described herein, and a polynucleotidecomprising the sequence of interest, wherein the engineered meganucleaseproduces a cleavage site in the dystrophin gene at a recognitionsequence comprising SEQ ID NO: 12, and wherein the sequence of interestis inserted into the dystrophin gene at the cleavage site. In someembodiments, the polynucleotide comprises nucleic acid sequenceshomologous to nucleic acid sequences flanking the cleavage site and thesequence of interest is inserted at the cleavage site by homologousrecombination. In some embodiments, the eukaryotic cell is a mammaliancell. In some embodiments, the mammalian cell is a muscle cell. In someembodiments, the muscle cell is a muscle precursor cell (e.g., asatellite cell or stem cell), a skeletal muscle cell, or a cardiacmuscle cell. In some embodiments, the mammalian cell is a human cell. Insome embodiments, the polynucleotide is introduced into the eukaryoticcell by a lipid nanoparticle, an mRNA, or a recombinant virus (e.g., arecombinant AAV).

In another aspect, the invention provides a method for producing agenetically modified eukaryotic cell comprising a modified dystrophingene, the method comprising: introducing into a eukaryotic cell one ormore polynucleotides comprising a first nucleic acid sequence encoding afirst engineered nuclease and a second nucleic acid sequence encoding asecond engineered nuclease, wherein the first engineered nuclease bindsand cleaves a recognition sequence in the intron 5′ upstream of exon 45,and wherein the second engineered nuclease binds and cleaves arecognition sequence in the intron 3′ downstream of exon 55, wherein thefirst engineered nuclease and the second engineered nuclease areexpressed in the eukaryotic cell, wherein the first engineered nucleaseproduces a first cleavage site in the dystrophin gene at its recognitionsequence, wherein the second engineered nuclease produces a secondcleavage site in the dystrophin gene at its recognition sequence,wherein the first cleavage site and the second cleavage site havecomplementary overhangs, wherein the intervening genomic DNA between thefirst cleavage site and the second cleavage site is excised from thedystrophin gene, and wherein the dystrophin gene is annealed to generatethe modified dystrophin gene.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 10. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease areselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 1. In such embodiments, the firstcleavage site and second cleavage site have complementary 3′ overhangs.In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.81 (SEQ ID NO: 46), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.63 (SEQ ID NO: 45), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.195 (SEQ ID NO: 47), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.282 (SEQ ID NO: 48), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.349 (SEQ ID NO: 49), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49), or a variantthereof described herein.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 12. In such embodiments, thefirst cleavage site and second cleavage site have complementary 3′overhangs. In some embodiments, the first engineered meganuclease andthe second engineered meganuclease are selected from the combinations ofmeganucleases (and variants thereof described herein) provided in Table2. In some embodiments, the first engineered meganuclease is DMD19-20x.13 (SEQ ID NO: 36), or a variant thereof described herein, andthe second engineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53), ora variant thereof described herein. In some embodiments, the firstengineered meganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variantthereof described herein, and the second engineered meganuclease is DMD37-38x.15 (SEQ ID NO: 53), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.66 (SEQ ID NO: 54), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.79 (SEQ ID NO: 55), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variantthereof described herein.

In some embodiments, the complementary overhangs (e.g., 3′ overhangs) ofthe first cleavage site and the second cleavage site are perfectlyligated to one another.

In some embodiments, the dystrophin gene comprises a nucleic acidsequence set forth in SEQ ID NO: 32 or 34. In some embodiments, thedystrophin gene comprises a nucleic acid sequence set forth in SEQ IDNO: 32. In some embodiments, the dystrophin gene comprises a nucleicacid sequence set forth in SEQ ID NO: 34.

In some embodiments, a normal reading frame is restored in the modifieddystrophin gene is restored as compared to a full-length wild-typedystrophin gene.

In some embodiments, the modified dystrophin gene encodes a modifieddystrophin polypeptide lacking the amino acids encoded by exons 45-55 ofa wild-type dystrophin gene. In some embodiments, the modifieddystrophin polypeptide comprises an amino acid sequence set forth in SEQID NO: 5.

In some embodiments, the method comprises introducing into theeukaryotic cell a first polynucleotide comprising a first nucleic acidsequence encoding the first engineered meganuclease and a secondpolynucleotide comprising a second nucleic acid sequence encoding thesecond engineered meganuclease. In some embodiments, the firstpolynucleotide is a first mRNA. In some embodiments, the secondpolynucleotide is a second mRNA. In some embodiments, the first mRNAand/or the second mRNA is an mRNA described herein (i.e., encoding anengineered meganuclease described herein). In some embodiments, thefirst polynucleotide is a first recombinant DNA construct. In someembodiments, the second polynucleotide is a second recombinant DNAconstruct. In some embodiments, the first recombinant DNA constructand/or the second recombinant DNA construct is a recombinant DNAconstruct described herein (i.e., comprising a nucleic acid sequenceencoding an engineered meganuclease described herein). In someembodiments, the first polynucleotide and the second polynucleotide areintroduced into the eukaryotic cell by one or more lipid nanoparticles.In some embodiments, the first polynucleotide is introduced into theeukaryotic cell by a first lipid nanoparticle. In some embodiments, thesecond polynucleotide is introduced into the eukaryotic cell by a secondlipid nanoparticle. In some embodiments, the first polynucleotide isintroduced into the eukaryotic cell by a first recombinant virus. Insome embodiments, the second polynucleotide is introduced into theeukaryotic cell by a second recombinant virus. In some embodiments, thefirst recombinant and/or the second recombinant virus are a recombinantvirus described herein (i.e., comprising a polynucleotide comprising anucleic acid sequence encoding an engineered meganuclease describedherein).

In some embodiments, the method comprises introducing into theeukaryotic cell a polynucleotide comprising a first nucleic acidsequence encoding the first engineered meganuclease and a second nucleicacid sequence encoding the second engineered meganuclease. In someembodiments, the polynucleotide is an mRNA. In some embodiments, themRNA is an mRNA described herein (i.e., comprising first and secondnucleic acid sequences each encoding meganucleases described herein). Insome embodiments, the polynucleotide is a recombinant DNA construct. Insome embodiments, the recombinant DNA construct is a recombinant DNAconstruct described herein (i.e., comprising first and second nucleicacid sequences each encoding meganucleases described herein). In someembodiments, the polynucleotide is introduced into the eukaryotic cellby a lipid nanoparticle. In some embodiments, the polynucleotide isintroduced into the eukaryotic cell by a recombinant virus. In someembodiments, the recombinant virus is a recombinant virus describedherein (i.e., comprising a polynucleotide comprising first and secondnucleic acid sequences each encoding meganucleases described herein).

In some embodiments, the eukaryotic cell is a mammalian cell. In someembodiments, the mammalian cell is a muscle cell. In some embodiments,the muscle cell is a muscle precursor cell (e.g., a satellite cell orstem cell), a skeletal muscle cell, or a cardiac muscle cell. In someembodiments, the mammalian cell is a human cell.

In another aspect, the invention provides a method for modifying adystrophin gene in a target cell in a subject, wherein the dystrophingene is characterized by a mutation that alters the reading frame of thedystrophin gene from wild-type, the method comprising: delivering to thetarget cell one or more polynucleotides comprising a first nucleic acidsequence encoding a first engineered nuclease and a second nucleic acidsequence encoding a second engineered nuclease, wherein the firstengineered nuclease binds and cleaves a recognition sequence in theintron 5′ upstream of exon 45, and wherein the second engineerednuclease binds and cleaves a recognition sequence in the intron 3′downstream of exon 55, wherein the first engineered nuclease and thesecond engineered nuclease are expressed in the target cell, wherein thefirst engineered nuclease produces a first cleavage site in thedystrophin gene at its recognition sequence, wherein the secondengineered nuclease produces a second cleavage site in the dystrophingene at its recognition sequence, wherein the first cleavage site andthe second cleavage site have complementary overhangs, wherein theintervening genomic DNA between the first cleavage site and the secondcleavage site is excised from the dystrophin gene, wherein thedystrophin gene is annealed, and wherein a normal reading frame of thedystrophin gene is restored as compared to a full-length wild-typedystrophin gene.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 10. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease areselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 1. In such embodiments, the firstcleavage site and second cleavage site have complementary 3′ overhangs.In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.81 (SEQ ID NO: 46), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.63 (SEQ ID NO: 45), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.195 (SEQ ID NO: 47), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.282 (SEQ ID NO: 48), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.349 (SEQ ID NO: 49), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49), or a variantthereof described herein.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 12. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease areselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 2. In such embodiments, the firstcleavage site and second cleavage site have complementary 3′ overhangs.In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.15 (SEQ ID NO: 53), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.66 (SEQ ID NO: 54), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.79 (SEQ ID NO: 55), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variantthereof described herein.

In some embodiments, the complementary overhangs (e.g., 3′ overhangs) ofthe first cleavage site and the second cleavage site are perfectlyligated to one another.

In some embodiments, the dystrophin gene comprises a nucleic acidsequence set forth in SEQ ID NO: 32 or 34. In some embodiments, thedystrophin gene comprises a nucleic acid sequence set forth in SEQ IDNO: 32. In some embodiments, the dystrophin gene comprises a nucleicacid sequence set forth in SEQ ID NO: 34.

In some embodiments, the dystrophin gene encodes a modified dystrophinpolypeptide lacking the amino acids encoded by exons 45-55 of awild-type dystrophin gene. In some embodiments, the modified dystrophinpolypeptide comprises an amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the subject is converted to a Becker MuscularDystrophy phenotype.

In some embodiments, the method comprises delivering to the target cella first polynucleotide comprising a first nucleic acid encoding thefirst engineered meganuclease and a second polynucleotide comprising asecond nucleic acid sequence encoding the second engineeredmeganuclease. In some embodiments, the first polynucleotide is a firstmRNA. In some embodiments, the second polynucleotide is a second mRNA.In some embodiments, the first mRNA and/or the second mRNA is adescribed herein (i.e., encoding an engineered meganuclease describedherein). In some embodiments, the first polynucleotide is a firstrecombinant DNA construct. In some embodiments, the secondpolynucleotide is a second recombinant DNA construct. In someembodiments, the first recombinant DNA construct and/or the secondrecombinant DNA construct is a recombinant DNA construct describedherein (i.e., comprising a nucleic acid sequence encoding an engineeredmeganuclease described herein). In some embodiments, the firstpolynucleotide and the second polynucleotide are delivered to the targetcells by one or more lipid nanoparticles. In some embodiments, the firstpolynucleotide is delivered to the target cell by a first lipidnanoparticle. In some embodiments, the second polynucleotide isdelivered to the target cell by a second lipid nanoparticle. In someembodiments, the first polynucleotide is delivered to the target cell bya first recombinant virus. In some embodiments, the secondpolynucleotide is delivered to the target cell by a second recombinantvirus. In some embodiments, the first recombinant and/or the secondrecombinant virus are a recombinant virus described herein (i.e.,comprising a polynucleotide comprising a nucleic acid sequence encodingan engineered meganuclease described herein).

In some embodiments, the method comprises delivering to the target cella polynucleotide comprising a first nucleic acid encoding the firstengineered meganuclease and a second nucleic acid sequence encoding thesecond engineered meganuclease. In some embodiments, the polynucleotideis an mRNA. In some embodiments, the mRNA is an mRNA described herein(i.e., comprising first and second nucleic acid sequences each encodingmeganucleases described herein). In some embodiments, the polynucleotideis a recombinant DNA construct. In some embodiments, the recombinant DNAconstruct is a recombinant DNA construct described herein (i.e.,comprising first and second nucleic acid sequences each encodingmeganucleases described herein). In some embodiments, the polynucleotideis delivered to the target cell by a lipid nanoparticle. In someembodiments, the polynucleotide is delivered to the target cell by arecombinant virus. In some embodiments, the recombinant virus is arecombinant virus described herein (i.e., comprising a polynucleotidecomprising first and second nucleic acid sequences each encodingmeganucleases described herein).

In some embodiments, the subject is a mammal. In some embodiments, thetarget cell is a muscle cell. In some embodiments, the muscle cell is amuscle precursor cell (e.g., a satellite cell or stem cell), a skeletalmuscle cell, or a cardiac muscle cell. In some embodiments, the subjectis a human.

In another aspect, the invention provides a method for treating DMD in asubject in need thereof, wherein the DMD is characterized by a mutationin a dystrophin gene that alters the reading frame of the dystrophingene relative to a full-length, wild-type dystrophin gene, the methodcomprising: administering to the subject an effective amount of one ormore polynucleotides comprising a first nucleic acid sequence encoding afirst engineered nuclease and a second nucleic acid sequence encoding asecond engineered nuclease, wherein the first engineered nuclease bindsand cleaves a recognition sequence in the intron 5′ upstream of exon 45,and wherein the second engineered nuclease binds and cleaves arecognition sequence in the intron 3′ downstream of exon 55, wherein theone or more polynucleotides are delivered to a target cell in thesubject, wherein the first engineered nuclease and the second engineerednuclease are expressed in the target cell, wherein the first engineerednuclease produces a first cleavage site in the dystrophin gene at itsrecognition sequence, wherein the second engineered nuclease produces asecond cleavage site in the dystrophin gene at its recognition sequence,wherein the first cleavage site and the second cleavage site havecomplementary overhangs, wherein the intervening genomic DNA between thefirst cleavage site and the second cleavage site is excised from thedystrophin gene, wherein the dystrophin gene is annealed, and wherein anormal reading frame of the dystrophin gene is restored as compared to afull-length wild-type dystrophin gene.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 10. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease areselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 1. In such embodiments, the firstcleavage site and second cleavage site have complementary 3′ overhangs.In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.63 (SEQ ID NO: 45), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.81 (SEQ ID NO: 46), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36x.81 (SEQ ID NO: 46), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36x.63 (SEQ ID NO: 45), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.195 (SEQ ID NO: 47), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.282 (SEQ ID NO: 48), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.282 (SEQ ID NO: 48), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20L.302 (SEQ ID NO: 39), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD35-36L.349 (SEQ ID NO: 49), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.329(SEQ ID NO: 40), or a variant thereof described herein, and the secondengineered meganuclease is DMD 35-36L.349 (SEQ ID NO: 49), or a variantthereof described herein.

In some embodiments, the first engineered nuclease is an engineeredmeganuclease described herein that binds and cleaves a recognitionsequence comprising SEQ ID NO: 6, and the second engineered nuclease isan engineered meganuclease described herein that binds and cleaves arecognition sequence comprising SEQ ID NO: 12. In some embodiments, thefirst engineered meganuclease and the second engineered meganuclease armselected from the combinations of meganucleases (and variants thereofdescribed herein) provided in Table 2. In such embodiments, the firstcleavage site and second cleavage site have complementary 3′ overhangs.In some embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.15 (SEQ ID NO: 53), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.15 (SEQ ID NO: 53), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.66 (SEQ ID NO: 54), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ TD NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.66 (SEQ ID NO: 54), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20x.13(SEQ ID NO: 36), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38x.79 (SEQ ID NO: 55), or a variantthereof described herein. In some embodiments, the first engineeredmeganuclease is DMD 19-20x.87 (SEQ ID NO: 37), or a variant thereofdescribed herein, and the second engineered meganuclease is DMD37-38x.79 (SEQ ID NO: 55), or a variant thereof described herein. Insome embodiments, the first engineered meganuclease is DMD 19-20L.249(SEQ ID NO: 38), or a variant thereof described herein, and the secondengineered meganuclease is DMD 37-38L.166 (SEQ ID NO: 56), or a variantthereof described herein.

In some embodiments, the complementary overhangs (e.g., 3′ overhangs) ofthe first cleavage site and the second cleavage site are perfectlyligated to one another.

In some embodiments, the dystrophin gene comprises a nucleic acidsequence set forth in SEQ ID NO: 32 or 34. In some embodiments, thedystrophin gene comprises a nucleic acid sequence set forth in SEQ IDNO: 32. In some embodiments, the dystrophin gene comprises a nucleicacid sequence set forth in SEQ ID NO: 34.

In some embodiments, the dystrophin gene encodes a modified dystrophinpolypeptide lacking the amino acids encoded by exons 45-55 of awild-type dystrophin gene. In some embodiments, the modified dystrophinpolypeptide comprises an amino acid sequence set forth in SEQ ID NO: 5.In some embodiments, the subject is converted to a Becker MuscularDystrophy phenotype.

In some embodiments, the method comprises administering to the subject afirst polynucleotide comprising a first nucleic acid encoding the firstengineered meganuclease and a second polynucleotide comprising a secondnucleic acid sequence encoding the second engineered meganuclease. Insome embodiments, the first polynucleotide is a first mRNA. In someembodiments, the second polynucleotide is a second mRNA. In someembodiments, the first mRNA and/or the second mRNA is a mRNA describedherein (i.e., encoding an engineered meganuclease described herein). Insome embodiments, the first polynucleotide is a first recombinant DNAconstruct. In some embodiments, the second polynucleotide is a secondrecombinant DNA construct. In some embodiments, the first recombinantDNA construct and/or the second recombinant DNA construct is arecombinant DNA construct described herein (i.e., comprising a nucleicacid sequence encoding an engineered meganuclease described herein). Insome embodiments, the first polynucleotide and the second polynucleotideare administered to the subject by a lipid nanoparticle. In someembodiments, the first polynucleotide is administered to the subject bya first lipid nanoparticle. In some embodiments, the secondpolynucleotide is administered to the subject by a second lipidnanoparticle. In some embodiments, the first polynucleotide isadministered to the subject by a first recombinant virus. In someembodiments, the second polynucleotide is administered to the subject bya second recombinant virus. In some embodiments, the first recombinantand/or the second recombinant virus are a recombinant virus describedherein (i.e., comprising a polynucleotide comprising a nucleic acidsequence encoding an engineered meganuclease described herein).

In some embodiments, the method comprises administering to the subject apolynucleotide comprising a first nucleic acid encoding the firstengineered meganuclease and a second nucleic acid sequence encoding thesecond engineered meganuclease. In some embodiments, the polynucleotideis an mRNA. In some embodiments, the mRNA is an mRNA described herein(i.e., comprising first and second nucleic acid sequences each encodingmeganucleases described herein). In some embodiments, the polynucleotideis a recombinant DNA construct. In some embodiments, the recombinant DNAconstruct is a recombinant DNA construct described herein (i.e.,comprising first and second nucleic acid sequences each encodingmeganucleases described herein). In some embodiments, the polynucleotideis administered to the subject by a lipid nanoparticle. In someembodiments, the polynucleotide is administered to the subject by arecombinant virus. In some embodiments, the recombinant virus is arecombinant virus described herein (i.e., comprising a polynucleotidecomprising first and second nucleic acid sequences each encodingmeganucleases described herein).

In some embodiments, the subject is a mammal. In some embodiments, thetarget cell is a muscle cell. In some embodiments, the muscle cell is amuscle precursor cell (e.g., a satellite cell or stem cell), a skeletalmuscle cell, or a cardiac muscle cell. In some embodiments, the subjectis a human.

In another aspect, the invention provides a polynucleotide comprising anucleic acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 34.

In some embodiments, the polynucleotide comprises a nucleic acidsequence set forth in SEQ ID NO: 32. In some embodiments, thepolynucleotide is a dystrophin gene in the genome of a cell (e.g., ahuman muscle cell) that comprises a nucleic acid sequence set forth inSEQ ID NO: 32. In some embodiments, the polynucleotide is a precursormRNA in a cell (e.g., a human muscle cell) that comprises a nucleic acidsequence set forth in SEQ ID NO: 32.

In some embodiments, the polynucleotide comprises a nucleic acidsequence set forth in SEQ ID NO: 34. In some embodiments, thepolynucleotide is a dystrophin gene in the genome of a cell (e.g., ahuman muscle cell) that comprises a nucleic acid sequence set forth inSEQ ID NO: 34. In some embodiments, the polynucleotide is a precursormRNA in a cell (e.g., a human muscle cell) that comprises a nucleic acidsequence set forth in SEQ ID NO: 34.

In another aspect, the invention provides a genetically modifiedeukaryotic cell comprising in its genome a modified dystrophin gene,wherein the modified dystrophin gene lacks exons 45-55, and wherein themodified dystrophin gene comprises a nucleic acid sequence set forth inSEQ ID NO: 32 or a nucleic acid sequence set forth in SEQ ID NO: 34positioned within an intron between exon 44 and exon 56.

In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 32.In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 34.

In some embodiments, the genetically modified eukaryotic cell is amammalian cell. In some embodiments, the genetically modified eukaryoticcell is a human cell. In some embodiments, the genetically modifiedeukaryotic cell is a muscle cell. In some embodiments, the muscle cellis a muscle precursor cell (e.g., a satellite cell or stem cell), askeletal muscle cell, or a cardiac muscle cell.

In another aspect, the invention provides a polypeptide comprising anamino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 5,wherein said polypeptide is a modified dystrophin protein lacking theamino acids encoded by exons 45-55 of the dystrophin gene, and whereinsaid polypeptide comprises the C-terminal domain of the dystrophinprotein. In some embodiments, the polypeptide comprises an amino acidsequence set forth in SEQ TD NO: 5.

In another aspect, the invention provides engineered meganucleasesdescribed herein, or polynucleotides described herein encodingengineered meganucleases, or cells described herein expressingengineered meganucleases, for use as a medicament.

In some embodiments, the medicament is useful for producing a modifieddystrophin gene in a subject. In some embodiments, the medicament isuseful for the treatment of DMD.

In another aspect, the invention provides the use of engineeredmeganucleases described herein, or polynucleotides disclosed hereinencoding engineered meganucleases, or cells described herein expressingengineered meganucleases, in the manufacture of a medicament fortreating DMD, for increasing levels of a modified dystrophin protein(i.e., lacking the amino acids encoded by exons 45-55 of the dystrophingene), or reducing the symptoms associated with DMD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic providing the approximate location of the DMDmeganuclease recognition sequences and illustrating the dualmeganuclease approach for excising multiple exons from the dystrophingene. As shown, pairs of engineered meganucleases that bind and cleaveeither the DMD 19-20 recognition sequence and the DMD 35-36 recognitionsequence, or the DMD 19-20 recognition sequence and the DMD 37-38recognition sequence, result in removal of exons 45-55 from thedystrophin gene. These pairs of recognition sequences are located withinintrons, have identical four basepair center sequences, and producecleavage sites having complementary overhangs. Therefore, following theremoval of the exons, the gene is ligated at the cleavage sites, whichwill be within an intron located between exon 44 and exon 56. Followingpost-transcriptional splicing, this genetic modification results in adystrophin mRNA having exon 44 in frame with exon 56, which restores thedystrophin gene reading frame and results in a Becker type of dystrophinexpression. Also shown is the approximate location where a pair ofengineered meganucleases bind and cleave the DMD 19-20 recognitionsequence and the DMD 29-30 recognition sequence, which results inremoval of exon 45 from the dystrophin gene.

FIG. 2 is a schematic showing exemplary recognition sequences of thedisclosure, which include sense and anti-sense sequences for the DMD19-20 (SEQ ID NOs: 6 and 7), DMD 29-30 (SEQ ID NOs: 8 and 9), DMD 35-36(SEQ ID NOs: 10 and 11), and DMD 37-38 (SEQ ID NOs: 12 and 13)recognition sequences in the human dystrophin gene. Each DMD recognitionsequence targeted by engineered meganucleases described herein comprisestwo recognition half-sites. Each recognition half-site comprises 9 basepairs, separated by a 4 basepair central sequence. For example, the DMD19-20 recognition sequence has a 5′ DMD19 half-site and a 3′ DMD20half-site with a four base pair center sequence GTAT.

FIG. 3 . The engineered meganucleases described herein comprise twosubunits, wherein the first subunit comprising the HVR1 region binds toa first recognition half-site (e.g., DMD19) and the second subunitcomprising the HVR2 region binds to a second recognition half-site(e.g., DMD20). In embodiments where the engineered meganuclease is asingle-chain meganuclease, the first subunit comprising the HVR1 regioncan be positioned as either the N-terminal or C-terminal subunit.Likewise, the second subunit comprising the HVR2 region can bepositioned as either the N-terminal or C-terminal subunit.

FIGS. 4A-4C. FIG. 4A provides an alignment of the sequences of DMD 19-20meganucleases. FIG. 4B provides an alignment of the sequences of DMD35-36 engineered meganucleases. FIG. 4C provides an alignment of thesequences of DMD 37-38 engineered meganucleases. Asterisks indicateconserved residues amongst all aligned nucleases, and a space indicatesthat at least one amino acid differed amongst the meganucleases.

FIG. 5 is a schematic of a reporter assay in CHO cells for evaluatingengineered meganucleases targeting recognition sequences found in thedystrophin gene. For the engineered meganucleases described herein, aCHO cell line was produced in which a reporter cassette was integratedstably into the genome of the cell. The reporter cassette comprised, in5′ to 3′ order: an SV40 Early Promoter; the 5′ 2/3 of the GFP gene; therecognition sequence for an engineered meganuclease described herein(e.g., the DMD 19-20, DMD 29-30, DMD 35-36, or DMD 37-38 recognitionsequences); the recognition sequence for the CHO-23/24 meganuclease (WO2012/167192); and the 3′ 2/3 of the GFP gene. Cells stably transfectedwith this cassette did not express GFP in the absence of a DNAbreak-inducing agent. Meganucleases were introduced by transduction ofan mRNA encoding each meganuclease. When a DNA break was induced ateither of the meganuclease recognition sequences, the duplicated regionsof the GFP gene recombined with one another to produce a functional GFPgene. The percentage of GFP-expressing cells could then be determined byflow cytometry as an indirect measure of the frequency of genomecleavage by the meganucleases.

FIGS. 6A-6F. FIGS. 6A, 6B, and 6G provide the efficiency of engineeredDMD 19-20 meganucleases for binding and cleaving the DMD 19-20recognition sequence expressed in the CHO cell reporter assay. FIG. 6Cprovides the efficiency of engineered DMD 29-30 meganucleases forbinding and cleaving the DMD 29-30 recognition sequence expressed in theCHO cell reporter assay. FIG. 6D and FIG. 6H provide the efficiency ofengineered DMD 35-36 meganucleases for binding and cleaving the DMD35-36 recognition sequence expressed in the CHO cell reporter assay.FIG. 6E. FIG. 6F, and FIG. 6I provide the efficiency of engineered DMD37-38 meganucleases for binding and cleaving the DMD 37-38 recognitionsequence expressed in the CHO cell reporter assay. The relative activityindex represents % GFP positive cells for each cell line expressing thetest meganuclease normalized to the cell line expressing the CHO-23/24meganuclease accounting for the toxicity of the meganuclease.

FIGS. 7A-7D show bar graphs showing the percentage frequency ofinsertions and deletions (indel) of the tested meganucleases targetingthe indicated recognition sequences at two doses in MRC5 cells. Eachmeganuclease was tested at three time points (days 2, 5, and 8)following transfection of the meganuclease. FIG. 7A provides thepercentage of editing with the DMD 19-20 meganucleases. FIG. 7B providesthe percentage of editing with the DMD 35-36 meganucleases. FIG. 7Cprovides the percentage of editing with the DMD 37-38 meganucleases.FIG. 7D provides the percentage of editing with the DMD 29-30meganucleases.

FIGS. 8A and 8B provide PCR product and sequencing data for the perfectligation of the dystrophin gene following cleavage with a pair ofengineered meganucleases designed to bind and cleave the DMD 19-20 andDMD 35-36 recognition sequences. FIG. 8A is a gel image of the PCRproduct using primers specific to amplify ligation of the complementaryDMD 19-20 and DMD 35-36 recognition sequences. Lane 5 represents thecombination of the DMD 19-20x.13 and DMD 35-36x.63 meganucleases. Lane 6represents the combination of the DMD 19-20x.87 and DMD 35-36x.81meganucleases. Lane 7 represents the combination of the DMD 19-20x.13and DMD 35-36x.81 meganucleases. Lane 8 represents the combination ofthe DMD 19-20x.87 and DMD 35-36x.63 meganucleases. Lane M represents amock control. FIG. 8B provides representative sequencing data forperfect ligation of the DMD 19-20 and DMD 35-36 meganuclease recognitionsequences following cleavage and excision of the intervening genomicsequence.

FIGS. 9A and 9B provide PCR product and sequencing data for the perfectligation of the dystrophin gene following cleavage with a pair ofengineered meganucleases designed to bind and cleave the DMD 19-20 andDMD 37-38 recognition sequences. FIG. 8A is a gel image of the PCRproduct using primers specific to amplify ligation of the complementaryDMD 19-20 and DMD 37-38 recognition sequences. Lane 9 represents thecombination of the DMD 19-20x.13 and DMD 37-38x.15 meganucleases. Lane10 represents the combination of the DMD 19-20x.87 and DMD 37-38x.15meganucleases. Lane 11 represents the combination of the DMD 19-20x.13and DMD 37-38x.66 meganucleases. Lane 12 represents the combination ofthe DMD 19-20x.87 and DMD 37-38x.66 meganucleases. Lane 13 representsthe combination of the DMD 19-20x.13 and DMD 37-38x.79 meganucleases.Lane 14 represents the combination of the DMD 19-20x.87 and DMD37-38x.79 meganucleases. Lane M represents a mock control. FIG. 9Bprovides a representative sequencing data for perfect ligation of theDMD 19-20 and DMD 37-38 meganuclease recognition sequences followingcleavage and excision of the intervening genomic sequence.

FIG. 10 provides PCR product for the perfect ligation of the dystrophingene following cleavage with a pair of engineered meganucleases designedto bind and cleave the DMD 19-20 and DMD 29-30 recognition sequences.Shown is a gel image of the PCR product using primers specific toamplify ligation of the complementary DMD 19-20 and DMD 29-30recognition sequences. Lane 1 represents the combination of the DMD19-20x.13 and DMD 29-30x.18 meganucleases. Lane 2 represents thecombination of the DMD 19-20x.87 and DMD 29-30x.40 meganucleases. Lane 3represents the combination of the DMD 19-20x.13 and DMD 29-30x.40meganucleases. Lane 4 represents the combination of the DMD 19-20x.87and DMD 29-30x.18 meganucleases. Lane M represents a mock control.

FIG. 11 provides a schematic showing the approximate location of theforward and reverse primers and the probe for the reference and perfectligation primer sets used in the Digital droplet PCR (ddPCR) assay fordetecting ligation events following cleavage. The schematic exemplifiesthe DMD 19-20 and DMD 37-38 ligated recognition sequences.

FIGS. 12A-12C provide bar graphs showing the percentage of deletion ofexons 45-55, or exon 45 alone, as assessed by ddPCR. FIG. 12A providesexon deletion data with the combination of the DMD 19-20x.13 and DMD35-36x.63 meganucleases, the DMD 19-20x.87 and DMD 35-36x.81meganucleases, the DMD 19-20x.13 and DMD 35-36x.81 meganucleases, theDMD 19-20x.87 and DMD 35-36x.63 meganucleases, and a mock control. FIG.12B provides exon deletion data with the combination of the DMD19-20x.13 and DMD 37-38x.15 meganucleases, the DMD 19-20x.87 and DMD37-38x.15 meganucleases, the DMD 19-20x.13 and DMD 37-38x.66meganucleases, the DMD 19-20x.87 and DMD 37-38x.66 meganucleases, theDMD 19-20x.13 and DMD 37-38x.79 meganucleases, the DMD 19-20x.87 and DMD37-38x.79 meganucleases, and a mock control. FIG. 12C provides exondeletion data for exon 45 alone with the combination of the DMD19-20x.13 and DMD 29-30x.18 meganucleases, the DMD 19-20x.87 and DMD29-30x.40 meganucleases, the DMD 19-20x.13 and DMD 29-30x.40meganucleases, the DMD 19-20x.87 and DMD 29-30x.18 meganucleases, and amock control.

FIG. 13 provides a line graph of perfect ligation events assessed byddPCR with additional different combinations of engineeredmeganucleases.

FIG. 14 provides a bar graph showing the percentage of deletion of exons45-55 as assessed by ddPCR using the pair of DMD 19-20L.249 and DMD37-38L.166 engineered meganucleases in human skeletal muscle myoblasts(HSMM). The HSMM cells were transfected with either 20 ng, 40 ng, 80 ng,or 160 ng of mRNA encoding each engineered meganuclease.

FIG. 15 provides a bar graph showing the percentage of perfect ligationassessed by ddPCR using the pair of DMD 19-20L.249 and DMD 37-38L.166engineered meganucleases in an immortalized myoblast cell line from apatient having DMD (AB1098 cells). The AB1098 cells were transfectedwith either 10 ng, 20 ng, 40 ng, 80 ng, or 160 ng of mRNA encoding eachmeganuclease.

FIGS. 16A-16C provide protein expression data following treatment witheither 10 ng, 20 ng, 40 ng, 80 ng, or 160 ng of mRNA encoding each ofthe DMD 19-20L.249 and DMD 37-38L.166 engineered meganucleases, or amock control, in AB1098 cells. FIG. 16A is a graph showing dystrophinprotein expression following treatment with the combination ofengineered meganucleases or the mock control. FIG. 16B provides a gelimage of the shortened dystrophin protein band (i.e., lacking the aminoacids encoded by exons 45-55) at the correct size. FIG. 16C provides theamount of dystrophin protein relative to a reference vinculin gene.

FIG. 17 provides a bar graph showing RNA splicing of exon 44 to exon 56in AB1098 cell mRNA following transfection with 10 ng, 20 ng, 40 ng, 80ng, or 160 ng of mRNA encoding each of the DMD 19-20L.249 and DMD37-38L.166 engineered meganucleases, or a mock control.

FIG. 18 provides a bar graph showing the percentage of perfect ligationas assessed by ddPCR using the indicated pairs of engineeredmeganucleases in HSMM cells. The HSMM cells were transfected with 40 ngof mRNA encoding each engineered meganuclease.

FIG. 19 provides a schematic of the oligo capture assay utilized todetermine off-target effects of an engineered nuclease (e.g., anengineered meganuclease described herein). As shown, the integrationcassette or oligo anneals with a double stranded break in the genomethat may be due to engineered nuclease cleavage. The DNA is then shearedby sonication, adapters are ligated and PCR amplified followed bysequence analysis to determine location of the double strand break.

FIG. 20 provides a graph depicting results from an oligo(oligonucleotide) capture assay to identify off-target cutting inducedby the DMD 19-20x.13, DMD 19-20L.249, DMD 19-20L.329, DMD 19-20L.374,DMD 19-20L.375, DMD 19-20L.431, and DMD 19-20L.458 meganucleasestransfected in HEK 293 cells. The circled dots indicate the on-targetsite and the non-circled dots indicate off-target sites with the X axisrepresenting the number of sequencing reads for each detected off-targetsite. The shade of the dot indicates the number of base-pair mismatchesbetween the on target site and each of the detected off-target sites.The closer to the top of the row the dot is located, the lower thenumber of mismatches.

FIG. 21 provides a graph depicting results from an oligo capture assayto identify off-target cutting induced by the DMD 35-36x.63, DMD35-36L.195, DMD 35-36L.364, DMD 35-36L.372, DMD 35-36L.457, and DMD35-36L.469 meganucleases transfected in HEK 293 cells. The circled dotsindicate the on-target site and the non-circled dots indicate off-targetsites with the X axis representing the number of sequencing reads foreach off-target site. The shade and proximity to the top of the row ofthe dot indicates the number of base-pair mismatches between the ontarget site and each of the detected off-target sites.

FIG. 22 provides a bar graph showing the percentage (%) of totalligation of genomic DNA adjacent to exons 45-55 following cleavage ofthe DMD 19-20 and DMD 35-36 recognition sequences by each of the pairsof indicated engineered DMD 19-20 and DMD 35-36 meganucleases.

FIG. 23A provides a WES protein intensity read out for shortenedmodified dystrophin protein levels lacking exons 45-55 of the dystrophingene in AB1098 cells treated with combinations of the DMD 19-20 and DMD35-36 meganucleases. Lane 1 is the marker control; lane 2 is the heartpositive control for dystrophin protein; lane 3 is the blank control;lane 4 is the combination of the DMD 19-20L.374 and DMD 35-36L.376meganucleases; lane 5 is the combination of the DMD 19-20L.374 and DMD35-36L.457 meganucleases; lane 6 is the combination of the DMD19-20L.374 and DMD 35-36L.469 meganucleases; lane 7 is the combinationof the DMD 19-20L.375 and DMD 35-36L.376 meganucleases; lane 8 is thecombination of the DMD 19-20L.375 and DMD 35-36L.457 meganucleases; lane9 is the combination of the DMD 19-20L.375 and DMD 35-36L.469meganucleases; lane 10 is the combination of the DMD 19-20L.431 and DMD35-36L.376 meganucleases; lane 11 is the combination of the DMD19-20L.431 and DMD 35-36L.457 meganucleases; lane 12 is the combinationof the DMD 19-20L.431 and DMD 35-36L.469 meganucleases; lane 13 is thecombination of the DMD 19-20L.458 and DMD 35-36L.376 meganucleases; lane14 is the combination of the DMD 19-20L.458 and DMD 35-36L.457meganucleases; lane 15 is the combination of the DMD 19-20L.458 and DMD35-36L.469 meganucleases; and lane 16 is the mock AB1098 cell linecontrol that lacks expression of dystrophin protein. FIG. 23B provides abar graph showing the shortened modified dystrophin protein levels byWES analysis normalized to the vinculin loading control for each of thepairs of indicated engineered DMD 19-20 and DMD 35-36 meganucleases.

FIGS. 24A-24B. FIG. 24A provides a bar graph showing the percentage (%)of total ligation of genomic DNA adjacent to exons 45-55 followingcleavage of the DMD 19-20 and DMD 37-38 recognition sequences by each ofthe pairs of indicated engineered DMD 19-20 and DMD 37-38 meganucleases.FIG. 24B provides a bar graph showing the percentage (%) dystrophinrestoration for each of the pairs of indicated engineered DMD 19-20 andDMD 37-38 meganucleases compared to an equivalent load of murinequadricep muscle tissue lysate that was based on a standard curvegenerated from that tissue.

FIGS. 25A-25E provides bar graphs showing the percentage (%) of perfectligation of genomic DNA adjacent to exons 45-55 in muscle tissuesfollowing cleavage of the DMD 19-20 and DMD 37-38 recognition sequencesby the pair of DMD 19-20x.13 and DMD 37-38x.15 engineered meganucleasesutilizing different muscle-specific promoter combinations. FIG. 25Ashows the percent perfect ligation in quadricep tissue. FIG. 25B showsthe percent perfect ligation in heart tissue. FIG. 25C shows the percentperfect ligation in diaphragm tissue. FIG. 25D shows the percent perfectligation in soleus tissue. FIG. 25E shows the percent perfect ligationin liver tissue.

FIGS. 26A-26E provides bar graphs showing the percentage (%) of totalligation of genomic DNA adjacent to exons 45-55 in muscle tissuesfollowing cleavage of the DMD 19-20 and DMD 35-36 recognition sequencesby the pair of DMD 19-20L.329 and DMD 37-38L.219 engineeredmeganucleases. FIG. 26A shows the percent total ligation in quadriceptissue. FIG. 26B shows the percent total ligation in heart tissue. FIG.26C shows the percent total ligation in diaphragm tissue. FIG. 25D showsthe percent total ligation in soleus tissue. FIG. 26E shows the percenttotal ligation in liver tissue.

FIGS. 27A-27E provides bar graphs showing the percentage (%) of totalligation of genomic DNA adjacent to exons 45-55 in muscle tissuesfollowing cleavage of the DMD 19-20 and DMD 35-36 recognition sequenceby the pair of DMD 19-20x.13 and DMD 37-38x.15 engineered meganucleasesat two different dosage levels indicated by the total AAV amount (2×10¹²or 4×10¹²). FIG. 27A shows the percent total ligation in quadriceptissue. FIG. 27B shows the percent total ligation in heart tissue. FIG.27C shows the percent total ligation in diaphragm tissue. FIG. 27D showsthe percent total ligation in tibialis anterior (TA) tissue. FIG. 27Eshows the percent total ligation in liver tissue.

FIGS. 28A-28C provides a WES protein intensity read out for shortenedmodified dystrophin protein levels lacking exons 45-55 of the dystrophingene after treatment with the DMD 19-20x.13 and DMD 37-38x.15meganucleases. Lanes 1-6 of FIGS. 28A-28C represent a standard curve ofprotein band intensity of full length human dystrophin from a mouse thatexpresses human dystrophin; lanes 7-8 represent the protein bandintensity of shortened modified dystrophin from mice treated with thecombination of the DMD 19-20x.13 and DMD 37-38x.15 meganucleases at1×10⁴ VG/kg; lanes 9-10 represent the protein band intensity ofshortened modified dystrophin from mice treated with the combination ofthe DMD 19-20x.13 and DMD 37-38x.15 meganucleases at 2×10¹⁴ VG/kg; lanes11-12 represent mice treated with PBS and without a meganuclease. FIG.28A represents modified shortened dystrophin levels detected at theindicated dosages in heart tissue; FIG. 28B represents modifiedshortened dystrophin levels detected at the indicated dosages indiaphragm tissue; FIG. 28C represents modified shortened dystrophinlevels detected at the indicated dosages in quadricep tissue.

FIG. 29 provides a graph showing the percentage (%) of total ligation ofgenomic DNA adjacent to exons 45-55 in the quadricep, heart, anddiaphragm muscle tissues following cleavage of the DMD 19-20 and DMD35-36 recognitions sequence by the pair of DMD 19-20L.329 and DMD35-36L.349 engineered meganucleases.

FIGS. 30A-30C provides a WES protein intensity read out for shortenedmodified dystrophin protein levels lacking exons 45-55 of the dystrophingene after treatment with the DMD 19-20L.329 and DMD 35-36L.349meganucleases utilizing different muscle specific promoters. Lane 1 ofFIGS. 30A-30C represent the ladder; lanes 2-6 represent band intensityof full-length human dystrophin from a mouse that expresses humandystrophin; lane 7 represents band intensity of shortened modifieddystrophin in mice treated with the combination of the DMD 19-20L.329and DMD 35-36L.349 meganucleases at 1×10¹⁴ VG/kg under the control ofthe CK8 muscle-specific promoter; lane 8 represents band intensity ofshortened modified dystrophin in mice treated with the combination ofthe DMD 19-20L.329 and DMD 35-36L.349 meganucleases at 1×10¹⁴ VG/kgunder the control of the MHCK7 muscle-specific promoter; lane 9represents band intensity of shortened modified dystrophin in micetreated with the combination of the DMD 19-20L.329 and DMD 35-36L.349meganucleases at 1×10¹⁴ VG/kg where the DMD 19-20L.329 meganuclease isunder the control of the CK8 muscle-specific promoter and the DMD35-36L.349 meganuclease is under the control of the SPc5-12 musclespecific promoter; lanes 10-11 represent mice treated with PBS. FIG. 30Arepresents modified shortened dystrophin levels detected at theindicated dosages in heart tissue; FIG. 309B represents modifiedshortened dystrophin levels detected at the indicated dosages indiaphragm tissue; and FIG. 30C represents modified shortened dystrophinlevels detected at the indicated dosages in quadricep tissue.

FIG. 31 provides a bar graph showing the modified shortened dystrophinprotein levels by WES analysis normalized to the vinculin loadingcontrol for mice treated with the DMD 19-20L.329 and DMD 35-36L.349meganucleases or PBS in the quadricep (quad), heart, and diaphragmtissue.

FIG. 32 provides immunohistochemistry imaging of the quadricep (quad),heart, and diaphragm muscle tissue from mice treated with thecombination of the DMD 19-20L.329 and DMD 35-36L.349 meganucleases orPBS. Dark staining represents human dystrophin detection, which is onlyseen in mice treated with the combination of the meganucleases.

FIGS. 33A and 33B provides fluorescent immunohistochemistry imaging ofmurine quadricep tissue following treatment with either PBS or the DMD19-20L.329 and DMD 35-36L.349 pair of meganucleases delivered using anAAV9 capsid to hDMDdel52/mdx (hDMD) mice. FIG. 33A provides imaging ofPax7 expression in quadricep tissue from PBS-treated mice. The leftpanel is a control image that shows any background staining with primaryand secondary antibodies that detect meganuclease expression. The middlepanel shows cells that express Pax7 indicated by the white arrow heads;and the right panel shows both Pax7 (white arrow heads) and anybackground staining from antibodies that detect meganuclease expression.FIG. 33B provides imaging of meganuclease and Pax7 expression inquadricep tissue from meganuclease treated mice. The left panel providesmeganuclease only expressing cells indicated by the white arrow headswith the full arrow indicating a cell that expresses meganucleaseprotein and Pax7; the middle panel shows cells that express Pax7 onlyindicated by the white arrowhead or Pax7 and meganuclease proteinindicated by the full arrow. The right panel shows cells that expresseither meganuclease protein or Pax7 indicated by the arrow heads or acell that expresses both meganuclease protein and Pax7 indicated by thefull arrow.

FIG. 34 provides a schematic of the BaseScope assay used to detect mRNAexpression of a modified dystrophin transcript where exons 45-55 of thehuman dystrophin gene have been deleted following expression of twonucleases. The first nuclease binds and cleaves a recognition sequencelocated in the intron immediately 5′ of exon 45, and the second nucleasebinds and cleaves a recognition sequence located in the intronimmediately 3′ of exon 55. As shown the nucleases that bind and cleaverecognition sequences located in these introns result in a double strandbreak that is then repaired by direct religation of the genome.Following transcription and splicing, an mRNA is produced with exon 44and exon 56 spliced together. A probe designed to recognize this exon 44to exon 56 junction (denoted as E44-E56 junction) is then used to detectthis modified human dystrophin transcript in muscle tissue sections.

FIGS. 35A and 35B provides the BaseScope staining of murine quadriceptissue from hDMDdel52/mdx (hDMD) mice that were treated with either PBSor the DMD 19-20L.329 and DMD 35-36L.349 pair of meganucleases. FIG. 35Ashows the BaseScope staining of muscle tissue from PBS treated mice forPax7 transcript expression and any background staining for the probeused to detect modified human dystrophin transcript expression. FIG. 35Bshows the BaseScope staining of muscle tissue from mice treated with theDMD 19-20L.329 and DMD 35-36L.349 pair of meganucleases for Pax7transcript expression and modified human dystrophin transcriptexpression.

FIG. 36 provides a WES protein intensity read out for shortened modifieddystrophin protein levels lacking exons 45-55 of the dystrophin geneafter electroporation of the KM1328 patient cell line that normallylacks dystrophin expression with increasing doses of the DMD 19-20L.329and DMD 35-36L.349 meganucleases at 20 ng, 80 ng, and 160 ng of mRNAencoding the meganucleases. Lane 1 represents the ladder, lane 2represents the mock control; lanes 3-5 represents the band intensity ofshortened modified dystrophin in cells treated with 20 ng, 80 ng, and160 ng of meganuclease mRNA; lane 6 represents the band intensity offull length human dystrophin.

FIG. 37 provides a WES protein intensity read out for shortened modifieddystrophin protein levels lacking exons 45-55 of the dystrophin geneafter electroporation of the AB1098 patient cell line that normallylacks dystrophin with the DMD 19-20L.329 and DMD 35-36L.349meganucleases at 80 ng of mRNA encoding the meganucleases. Lane 1represents the band intensity of full length human dystrophin; lane 2represents the mock control; lane 3 represents the band intensity ofshortened modified dystrophin in cells treated with 80 ng ofmeganuclease mRNA.

FIGS. 38A-38D provides fluorescent immunohistochemistry imaging ofmurine quadricep tissue following treatment with either PBS or the DMD19-20L.329 and DMD 35-36L.349 pair of meganucleases delivered using anAAVrH74 capsid to hDMDdel52/mdx (hDMD) mice. FIG. 38A provides imagingof Pax7 expression in quadricep tissue from PBS treated mice. The leftpanel is a control image that provides any background staining withprimary and secondary antibodies that detect meganuclease expression;the star indicates non-specific background detection. The middle panelshows cells that express Pax7 indicated by the white arrowhead; and theright panel shows both Pax7 (white arrowhead) and any backgroundstaining from antibodies that detect meganuclease expression indicatedby the star. FIG. 38B provides imaging of meganuclease and Pax7expression in quadricep tissue from meganuclease treated mice at adosage of 1×10¹⁴ VG/kg; FIG. 38C provides imaging from meganucleasetreated mice at a dosage of 3×10¹³VG/kg; and FIG. 38D provides imagingfrom meganuclease-treated mice at a dosage of 1×10¹³ VG/kg. The leftpanel in FIGS. 38B-38D provides meganuclease-only expressing cells; themiddle panel shows cells that express Pax7; and the right panel in FIGS.38B-38D shows cells that express either meganuclease protein or Pax7 orcells that expresses both meganuclease protein and Pax7 indicated by thefull arrow. Stars indicate non-specific background staining.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 sets forth amino acid sequence of the wild-type I-CreImeganuclease from Chlamydomonas reinhardtii.

SEQ ID NO: 2 sets forth the amino acid sequence of the LAGLIDADG motif.

SEQ ID NO: 3 sets forth the amino acid sequence of a nuclearlocalization signal.

SEQ ID NO: 4 sets forth the amino acid sequence of the wild-typedystrophin protein CCDS48091.1 (Gene ID 1756).

SEQ ID NO: 5 sets forth amino acid sequence of the wild-type dystrophinprotein CCDS48091.1 (Gene ID 1756) lacking amino acids encoded by exons45-55.

SEQ ID NO: 6 sets forth the nucleic acid sequence of the sense strand ofthe DMD 19-20 recognition sequence.

SEQ ID NO: 7 sets forth the nucleic acid sequence of the antisensestrand of the DMD 19-20 recognition sequence.

SEQ ID NO: 8 sets forth the nucleic acid sequence of the sense strand ofthe DMD 29-30 recognition sequence.

SEQ ID NO: 9 sets forth the nucleic acid sequence of the antisensestrand of the DMD 29-30 recognition sequence.

SEQ ID NO: 10 sets forth the nucleic acid sequence of the sense strandof the DMD 35-36 recognition sequence.

SEQ ID NO: 11 sets forth the nucleic acid sequence of the antisensestrand of the DMD 35-36 recognition sequence.

SEQ ID NO: 12 sets forth the nucleic acid sequence of the sense strandof the DMD 37-38 recognition sequence.

SEQ ID NO: 13 sets forth the nucleic acid sequence of the antisensestrand of the DMD 37-38 recognition sequence.

SEQ ID NO: 14 sets forth the nucleic acid sequence of the firsthalf-site sense strand of the DMD 19-20 recognition sequence.

SEQ ID NO: 15 sets forth the nucleic acid sequence of the firsthalf-site antisense strand of the DMD 19-20 recognition sequence.

SEQ ID NO: 16 sets forth the nucleic acid sequence of the firsthalf-site sense strand of the DMD 29-30 recognition sequence.

SEQ ID NO: 17 sets forth the nucleic acid sequence of the firsthalf-site antisense strand of the DMD 29-30 recognition sequence.

SEQ ID NO: 18 sets forth the nucleic acid sequence of the firsthalf-site sense strand of the DMD 35-36 recognition sequence.

SEQ ID NO: 19 sets forth the nucleic acid sequence of the firsthalf-site antisense strand of the DMD 35-36 recognition sequence.

SEQ ID NO: 20 sets forth the nucleic acid sequence of the firsthalf-site sense strand of the DMD 37-38 recognition sequence.

SEQ ID NO: 21 sets forth the nucleic acid sequence of the firsthalf-site antisense strand of the DMD 37-38 recognition sequence.

SEQ ID NO: 22 sets forth the nucleic acid sequence of the secondhalf-site sense strand of the DMD 19-20 recognition sequence.

SEQ ID NO: 23 sets forth the nucleic acid sequence of the secondhalf-site antisense strand of the DMD 19-20 recognition sequence.

SEQ ID NO: 24 sets forth the nucleic acid sequence of the secondhalf-site sense strand of the DMD 29-30 recognition sequence.

SEQ ID NO: 25 sets forth the nucleic acid sequence of the secondhalf-site antisense strand of the DMD 29-30 recognition sequence.

SEQ ID NO: 26 sets forth the nucleic acid sequence of the secondhalf-site sense strand of the DMD 35-36 recognition sequence.

SEQ ID NO: 27 sets forth the nucleic acid sequence of the secondhalf-site antisense strand of the DMD 35-36 recognition sequence.

SEQ ID NO: 28 sets forth the nucleic acid sequence of the secondhalf-site sense strand of the DMD 37-38 recognition sequence.

SEQ ID NO: 29 sets forth the nucleic acid sequence of the secondhalf-site antisense strand of the DMD 37-38 recognition sequence.

SEQ ID NO: 30 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/29-30 sense strands.

SEQ ID NO: 31 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/29-30 sense strands.

SEQ ID NO: 32 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/35-36 sense strands.

SEQ ID NO: 33 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/35-36 sense strands.

SEQ ID NO: 34 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/37-38 sense strands.

SEQ ID NO: 35 sets forth the nucleic acid sequence of the ligated hybridDMD 19-20/37-38 sense strands.

SEQ ID NO: 36 sets forth the amino acid sequence of the DMD 19-20x.13engineered meganuclease.

SEQ ID NO: 37 sets forth the amino acid sequence of the DMD 19-20x.87engineered meganuclease.

SEQ ID NO: 38 sets forth the amino acid sequence of the DMD 19-20L.249engineered meganuclease.

SEQ ID NO: 39 sets forth the amino acid sequence of the DMD 19-20L.302engineered meganuclease.

SEQ ID NO: 40 sets forth the amino acid sequence of the DMD 19-20L.329engineered meganuclease.

SEQ ID NO: 41 sets forth the amino acid sequence of the DMD 19-20L.374engineered meganuclease.

SEQ ID NO: 42 sets forth the amino acid sequence of the DMD 19-20L.375engineered meganuclease.

SEQ ID NO: 43 sets forth the amino acid sequence of the DMD 19-20L.431engineered meganuclease.

SEQ ID NO: 44 sets forth the amino acid sequence of the DMD 19-20L.458engineered meganuclease.

SEQ ID NO: 45 sets forth the amino acid sequence of the DMD 35-36x.63engineered meganuclease.

SEQ ID NO: 46 sets forth the amino acid sequence of the DMD 35-36x.81engineered meganuclease.

SEQ ID NO: 47 sets forth the amino acid sequence of the DMD 35-36L.195engineered meganuclease.

SEQ ID NO: 48 sets forth the amino acid sequence of the DMD35-36L.282engineered meganuclease.

SEQ ID NO: 49 sets forth the amino acid sequence of the DMD35-36L.349engineered meganuclease.

SEQ ID NO: 50 sets forth the amino acid sequence of the DMD 35-36L.376engineered meganuclease.

SEQ ID NO: 51 sets forth the amino acid sequence of the DMD 35-36L.457engineered meganuclease.

SEQ ID NO: 52 sets forth the amino acid sequence of the DMD 35-36L.469engineered meganuclease.

SEQ ID NO: 53 sets forth the amino acid sequence of the DMD 37-38x.15engineered meganuclease.

SEQ ID NO: 54 sets forth the amino acid sequence of the DMD 37-38x.66engineered meganuclease.

SEQ ID NO: 55 sets forth the amino acid sequence of the DMD 37-38x.79engineered meganuclease.

SEQ ID NO: 56 sets forth the amino acid sequence of the DMD 37-38L.166engineered meganuclease.

SEQ ID NO: 57 sets forth the amino acid sequence of the DMD 37-38L.478engineered meganuclease.

SEQ ID NO: 58 sets forth the amino acid sequence of the DMD 37-38L.512engineered meganuclease.

SEQ ID NO: 59 sets forth the amino acid sequence of the DMD 37-38L.528engineered meganuclease.

SEQ ID NO: 60 sets forth a nucleic acid sequence encoding the DMD19-20x.13 engineered meganuclease.

SEQ ID NO: 61 sets forth a nucleic acid sequence encoding the DMD19-20x.87 engineered meganuclease.

SEQ ID NO: 62 sets forth a nucleic acid sequence encoding the DMD19-20L.249 engineered meganuclease.

SEQ ID NO: 64 sets forth a nucleic acid sequence encoding the DMD19-20L.302 engineered meganuclease.

SEQ ID NO: 64 sets forth a nucleic acid sequence encoding the DMD19-20L.329 engineered meganuclease.

SEQ ID NO: 65 sets forth the nucleic acid sequence of the DMD 19-20L.374engineered meganuclease.

SEQ ID NO: 66 sets forth the nucleic acid sequence of the DMD 19-20L.375engineered meganuclease.

SEQ ID NO: 67 sets forth the nucleic acid sequence of the DMD 19-20L.431engineered meganuclease.

SEQ ID NO: 68 sets forth the nucleic acid sequence of the DMD 19-20L.458engineered meganuclease.

SEQ ID NO: 69 sets forth a nucleic acid sequence encoding the DMD35-36x.63 engineered meganuclease.

SEQ ID NO: 70 sets forth a nucleic acid sequence encoding the DMD35-36x.81 engineered meganuclease.

SEQ ID NO: 71 sets forth a nucleic acid sequence encoding the DMD35-36L.195 engineered meganuclease.

SEQ ID NO: 72 sets forth a nucleic acid sequence encoding theDMD35-36L.282 engineered meganuclease.

SEQ ID NO: 73 sets forth a nucleic acid sequence encoding theDMD35-36L.349 engineered meganuclease.

SEQ ID NO: 74 sets forth a nucleic acid sequence encoding theDMD35-36L.376 engineered meganuclease.

SEQ ID NO: 75 sets forth a nucleic acid sequence encoding theDMD35-36L.457 engineered meganuclease.

SEQ ID NO: 76 sets forth a nucleic acid sequence encoding theDMD35-36L.469 engineered meganuclease.

SEQ ID NO: 77 sets forth a nucleic acid sequence encoding the DMD37-38x.15 engineered meganuclease.

SEQ ID NO: 78 sets forth a nucleic acid sequence encoding the DMD37-38x.66 engineered meganuclease.

SEQ ID NO: 79 sets forth a nucleic acid sequence encoding the DMD37-38x.79 engineered meganuclease.

SEQ ID NO: 80 sets forth a nucleic acid sequence encoding the DMD37-38L.166 engineered meganuclease.

SEQ ID NO: 81 sets forth a nucleic acid sequence encoding the DMD37-38L.478 engineered meganuclease.

SEQ ID NO: 82 sets forth a nucleic acid sequence encoding the DMD37-38L.512 engineered meganuclease.

SEQ ID NO: 83 sets forth a nucleic acid sequence encoding the DMD37-38L.528 engineered meganuclease.

SEQ ID NO: 84 sets forth the amino acid sequence of the DMD 19-20x.13engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 85 sets forth the amino acid sequence of the DMD 19-20x.87engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 86 sets forth the amino acid sequence of the DMD 19-20L.249engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 87 sets forth the amino acid sequence of the DMD 19-20L.302engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 88 sets forth the amino acid sequence of the DMD 19-20L.329engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 89 sets forth the amino acid sequence of the DMD 19-20L.374engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 90 sets forth the amino acid sequence of the DMD 19-20L.375engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 91 sets forth the amino acid sequence of the DMD 19-20L.431engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 92 sets forth the amino acid sequence of the DMD 19-20L.458engineered meganuclease DMD19 binding subunit.

SEQ ID NO: 93 sets forth the amino acid sequence of the DMD 35-36x.63engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 94 sets forth the amino acid sequence of the DMD 35-36x.81engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 95 sets forth the amino acid sequence of the DMD 35-36L.195engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 96 sets forth the amino acid sequence of the DMD35-36L.282engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 97 sets forth the amino acid sequence of the DMD35-36L.349engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 98 sets forth the amino acid sequence of the DMD35-36L.376engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 99 sets forth the amino acid sequence of the DMD35-36L.457engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 100 sets forth the amino acid sequence of the DMD35-36L.469engineered meganuclease DMD35 binding subunit.

SEQ ID NO: 101 sets forth the amino acid sequence of the DMD 37-38x.15engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 102 sets forth the amino acid sequence of the DMD 37-38x.66engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 103 sets forth the amino acid sequence of the DMD 37-38x.79engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 104 sets forth the amino acid sequence of the DMD 37-38L.166engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 105 sets forth the amino acid sequence of the DMD 37-38L.478engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 106 sets forth the amino acid sequence of the DMD 37-38L.512engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 107 sets forth the amino acid sequence of the DMD 37-38L.528engineered meganuclease DMD37 binding subunit.

SEQ ID NO: 108 sets forth the amino acid sequence of the DMD 19-20x.13engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 109 sets forth the amino acid sequence of the DMD 19-20x.87engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 110 sets forth the amino acid sequence of the DMD 19-20L.249engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 111 sets forth the amino acid sequence of the DMD 19-20L.302engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 112 sets forth the amino acid sequence of the DMD 19-20L.329engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 113 sets forth the amino acid sequence of the DMD 19-20L.374engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 114 sets forth the amino acid sequence of the DMD 19-20L.375engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 115 sets forth the amino acid sequence of the DMD 19-20L.431engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 116 sets forth the amino acid sequence of the DMD 19-20L.458engineered meganuclease DMD20 binding subunit.

SEQ ID NO: 117 sets forth the amino acid sequence of the DMD 35-36x.63engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 118 sets forth the amino acid sequence of the DMD 35-36x.81engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 119 sets forth the amino acid sequence of the DMD 35-36L.195engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 120 sets forth the amino acid sequence of the DMD35-36L.282engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 121 sets forth the amino acid sequence of the DMD35-36L.349engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 122 sets forth the amino acid sequence of the DMD35-36L.376engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 123 sets forth the amino acid sequence of the DMD35-36L.457engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 124 sets forth the amino acid sequence of the DMD35-36L.469engineered meganuclease DMD36 binding subunit.

SEQ ID NO: 125 sets forth the amino acid sequence of the DMD 37-38x.15engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 126 sets forth the amino acid sequence of the DMD 37-38x.66engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 127 sets forth the amino acid sequence of the DMD 37-38x.79engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 128 sets forth the amino acid sequence of the DMD 37-38L.166engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 129 sets forth the amino acid sequence of the DMD 37-38L.478engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 130 sets forth the amino acid sequence of the DMD 37-38L.512engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 131 sets forth the amino acid sequence of the DMD 37-38L.528engineered meganuclease DMD38 binding subunit.

SEQ ID NO: 132 sets forth the amino acid sequence of a linker sequence.

SEQ ID NO: 133 sets forth the nucleic acid sequence of a probe used in addPCR assay for detecting INDELs at the DMD 19-20 recognition sequence.

SEQ ID NO: 134 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 19-20recognition sequence.

SEQ ID NO: 135 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 19-20recognition sequence.

SEQ ID NO: 136 sets forth the nucleic acid sequence of a probe used as areference in a ddPCR assay for detecting INDELs.

SEQ ID NO: 137 sets forth the nucleic acid sequence of a forward PCRprimer used as a reference in a ddPCR assay for detecting INDELs.

SEQ ID NO: 138 sets forth the nucleic acid sequence of a forward PCRprimer used as a reference in a ddPCR assay for detecting INDELs.

SEQ ID NO: 139 sets forth the nucleic acid sequence of a probe used in addPCR assay for detecting INDELs at the DMD 37-38 recognition sequence.

SEQ ID NO: 140 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 37-38recognition sequence.

SEQ ID NO: 141 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 37-38recognition sequence.

SEQ ID NO: 142 sets forth the nucleic acid sequence of a probe used in addPCR assay for detecting INDELs at the DMD 35-36 recognition sequence.

SEQ ID NO: 143 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 35-36recognition sequence.

SEQ ID NO: 144 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 35-36recognition sequence.

SEQ ID NO: 145 sets forth the nucleic acid sequence of a probe used in addPCR assay for detecting INDELs at the DMD 29-30 recognition sequence.

SEQ ID NO: 146 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 29-30recognition sequence.

SEQ ID NO: 147 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for detecting INDELs at the DMD 29-30recognition sequence.

SEQ ID NO: 148 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 35-36ligated recognition sequences.

SEQ ID NO: 149 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 35-36ligated recognition sequences.

SEQ ID NO: 150 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 35-36ligated recognition sequences.

SEQ ID NO: 151 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 35-36ligated recognition sequences.

SEQ ID NO: 152 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 29-30ligated recognition sequences.

SEQ ID NO: 153 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 29-30ligated recognition sequences.

SEQ ID NO: 154 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 29-30ligated recognition sequences.

SEQ ID NO: 155 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 29-30ligated recognition sequences.

SEQ ID NO: 156 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 37-38ligated recognition sequences.

SEQ ID NO: 157 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 37-38ligated recognition sequences.

SEQ ID NO: 158 sets forth the nucleic acid sequence of a forward PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 37-38ligated recognition sequences.

SEQ ID NO: 159 sets forth the nucleic acid sequence of a reverse PCRprimer used in a PCR amplification assay for the DMD 19-20 to DMD 37-38ligated recognition sequences.

SEQ ID NO: 160 sets forth the nucleic acid sequence of a probe used in addPCR assay for the DMD 19-20 to DMD 37-38 ligated recognitionsequences.

SEQ ID NO: 161 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 37-38 ligatedrecognition sequences.

SEQ ID NO: 162 sets forth the nucleic acid sequence of a reverse PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 37-38 ligatedrecognition sequences.

SEQ ID NO: 163 sets forth the nucleic acid sequence of a probe used in addPCR assay for the DMD 19-20 to DMD 35-36 ligated recognitionsequences.

SEQ ID NO: 164 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 35-36 ligatedrecognition sequences.

SEQ ID NO: 165 sets forth the nucleic acid sequence of a reverse PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 35-36 ligatedrecognition sequences.

SEQ ID NO: 166 sets forth the nucleic acid sequence of a probe used in addPCR assay for the DMD 19-20 to DMD 29-30 ligated recognitionsequences.

SEQ ID NO: 167 sets forth the nucleic acid sequence of a forward PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 29-30 ligatedrecognition sequences.

SEQ ID NO: 168 sets forth the nucleic acid sequence of a reverse PCRprimer used in a ddPCR assay for the DMD 19-20 to DMD 29-30 ligatedrecognition sequences.

SEQ ID NO: 169 sets forth the nucleic acid sequence of a C5-12 promotersequence.

SEQ ID NO: 170 sets forth the nucleic acid sequence of a murine MCKpromoter and enhancer sequence.

SEQ ID NO: 171 sets forth the nucleic acid sequence of a human MCKpromoter sequence.

SEQ ID NO: 172 sets forth the nucleic acid sequence of a wild-type MCKenhancer sequence.

SEQ ID NO: 173 sets forth the nucleic acid sequence of a modified MCKenhancer sequence.

SEQ ID NO: 174 sets forth the nucleic acid sequence of a spc 5-12promoter sequence.

SEQ ID NO: 175 sets forth the nucleic acid sequence of a MHCK7 promotersequence.

SEQ ID NO: 176 sets forth the nucleic acid sequence of a CK8 promotersequence.

SEQ ID NO: 177 sets forth the nucleic acid sequence of a SK-CRM4promoter sequence.

SEQ ID NO: 178 sets forth the nucleic acid sequence of a SP-301 promotersequence.

SEQ ID NO: 179 sets forth the nucleic acid sequence of a SP-817 promotersequence.

SEQ ID NO: 180 sets forth the nucleic acid sequence of a SP-905 promotersequence.

SEQ ID NO: 181 sets forth the nucleic acid sequence of a Muscle Hybridpromoter sequence.

SEQ ID NO: 182 sets forth the amino acid sequence of an rh.74 AAVcapsid.

SEQ ID NO: 183 sets forth the amino acid sequence of an AAV9 capsid.

SEQ ID NO: 184 sets forth the nucleic acid sequence of a forward primer.

SEQ ID NO: 185 sets forth the nucleic acid sequence of a reverse primer.

SEQ ID NO: 186 sets forth the nucleic acid sequence of a probe.

SEQ ID NO: 187 sets forth the nucleic acid sequence of a forward primer.

SEQ ID NO: 188 sets forth the nucleic acid sequence of a reverse primer.

SEQ ID NO: 189 sets forth the nucleic acid sequence of a probe.

SEQ ID NO: 190 sets forth the nucleic acid sequence of a forward primer.

SEQ ID NO: 191 sets forth the nucleic acid sequence of a reverse primer.

SEQ ID NO: 192 sets forth the nucleic acid sequence of a probe.

SEQ ID NO: 193 sets forth the nucleic acid sequence of a reverse primer.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present disclosure can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the present disclosure, which do not depart from thepresent invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

As used herein, the terms “nuclease” and “endonuclease” are usedinterchangeably to refer to naturally-occurring or engineered enzymes,which cleave a phosphodiester bond within a polynucleotide chain.Engineered nucleases can include, without limitation, engineeredmeganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPRsystem nucleases, and megaTALs. In addition, any engineered nuclease isenvisioned that is capable of generating overhangs at its cleavage site.

As used herein, the terms “cleave” or “cleavage” refer to the hydrolysisof phosphodiester bonds within the backbone of a recognition sequencewithin a target sequence that results in a double-stranded break withinthe target sequence, referred to herein as a “cleavage site”.

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. In some embodiments, the recognition sequence for ameganuclease of the present disclosure is 22 base pairs. A meganucleasecan be an endonuclease that is derived from I-CreI (SEQ ID NO: 1), andcan refer to an engineered variant of I-CreI that has been modifiedrelative to natural I-CreI with respect to, for example, DNA-bindingspecificity, DNA cleavage activity, DNA-binding affinity, ordimerization properties. Methods for producing such modified variants ofI-CreI are known in the art (e.g., WO 2007/047859, incorporated byreference in its entirety). A meganuclease as used herein binds todouble-stranded DNA as a heterodimer. A meganuclease may also be a“single-chain meganuclease” in which a pair of DNA-binding domains isjoined into a single polypeptide using a peptide linker. The term“homing endonuclease” is synonymous with the term “meganuclease.”Meganucleases of the present disclosure are substantially non-toxic whenexpressed in the targeted cells as described herein such that cells canbe transfected and maintained at 37° C. without observing deleteriouseffects on cell viability or significant reductions in meganucleasecleavage activity when measured using the methods described herein.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of nuclease subunits joined by a linker. Asingle-chain meganuclease has the organization: N-terminalsubunit—Linker—C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will bindnon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two nuclease subunits into a single polypeptide. Alinker may have a sequence that is found in natural proteins or may bean artificial sequence that is not found in any natural protein. Alinker may be flexible and lacking in secondary structure or may have apropensity to form a specific three-dimensional structure underphysiological conditions. A linker can include, without limitation,those encompassed by U.S. Pat. Nos. 8,445,251, 9,340,777, 9,434,931, and10,041,053, each of which is incorporated by reference in its entirety.In some embodiments, a linker may have at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 132, which sets forth residues154-195 of any one of SEQ ID NOs: 36-59.

As used herein, the terms “recombinant” or “engineered,” with respect toa protein, means having an altered amino acid sequence as a result ofthe application of genetic engineering techniques to nucleic acids thatencode the protein and cells or organisms that express the protein. Withrespect to a nucleic acid, the term “recombinant” or “engineered” meanshaving an altered nucleic acid sequence as a result of the applicationof genetic engineering techniques. Genetic engineering techniquesinclude, but are not limited to, PCR and DNA cloning technologies;transfection, transformation, and other gene transfer technologies;homologous recombination; site-directed mutagenesis; and gene fusion. Inaccordance with this definition, a protein having an amino acid sequenceidentical to a naturally-occurring protein, but produced by cloning andexpression in a heterologous host, is not considered recombinant orengineered.

As used herein, the term “wild-type” refers to the most common naturallyoccurring allele (i.e., polynucleotide sequence) in the allelepopulation of the same type of gene, wherein a polypeptide encoded bythe wild-type allele has its original functions. The term “wild-type”also refers to a polypeptide encoded by a wild-type allele. Wild-typealleles (i.e., polynucleotides) and polypeptides are distinguishablefrom mutant or variant alleles and polypeptides, which comprise one ormore mutations and/or substitutions relative to the wild-typesequence(s). Whereas a wild-type allele or polypeptide can confer anormal phenotype in an organism, a mutant or variant allele orpolypeptide can, in some instances, confer an altered phenotype.Wild-type nucleases are distinguishable from recombinant ornon-naturally-occurring nucleases. The term “wild-type” can also referto a cell, an organism, and/or a subject which possesses a wild-typeallele of a particular gene, or a cell, an organism, and/or a subjectused for comparative purposes.

As used herein, the term “genetically modified” refers to a cell ororganism in which, or in an ancestor of which, a genomic DNA sequencehas been deliberately modified by recombinant technology. As usedherein, the term “genetically modified” encompasses the term“transgenic.”

As used herein, the term with respect to recombinant proteins, the term“modification” means any insertion, deletion, or substitution of anamino acid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the terms “recognition sequence” or “recognition site”refers to a DNA sequence that is bound and cleaved by a nuclease. In thecase of a meganuclease, a recognition sequence comprises a pair ofinverted, 9 basepair “half-sites,” which are separated by fourbasepairs. In the case of a single-chain meganuclease, the N-terminaldomain of the protein contacts a first half-site and the C-terminaldomain of the protein contacts a second half-site. Cleavage by ameganuclease produces four basepair 3′ overhangs. “Overhangs,” or“sticky ends” are short, single-stranded DNA segments that can beproduced by endonuclease cleavage of a double-stranded DNA sequence. Inthe case of meganucleases and single-chain meganucleases derived fromI-CreI, the overhang comprises bases 10-13 of the 22 basepairrecognition sequence.

As used herein, the terms “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a nuclease.

As used herein, the terms “DNA-binding affinity” or “binding affinity”means the tendency of a nuclease to non-covalently associate with areference DNA molecule (e.g., a recognition sequence or an arbitrarysequence). Binding affinity is measured by a dissociation constant, Kd.As used herein, a nuclease has “altered” binding affinity if the Kd ofthe nuclease for a reference recognition sequence is increased ordecreased by a statistically significant percent change relative to areference nuclease.

As used herein, the term “specificity” means the ability of a nucleaseto bind and cleave double-stranded DNA molecules only at a particularsequence of base pairs referred to as the recognition sequence, or onlyat a particular set of recognition sequences. The set of recognitionsequences will share certain conserved positions or sequence motifs butmay be degenerate at one or more positions. A highly-specific nucleaseis capable of cleaving only one or a very few recognition sequences.Specificity can be determined by any method known in the art.

As used herein, the term “dystrophin gene” refers to the gene associatedwith National Center for Biotechnology Information (NCBI) gene ID 1756,as well as naturally occurring variants thereof. The term “dystrophin”refers to a polypeptide encoded by the dystrophin gene. The dystrophinisoform expressed in muscle cells and muscle precursor cells is known asthe Dp427m dystrophin variant. The amino acid sequence of a full-length,wild type Dp427m dystrophin polypeptide is set forth in SEQ ID NO: 4.NCBI reference numbers NM_004006.3 and NP_003997.2 set forth thedystrophin Dp427m mRNA and polypeptide, respectively. In someembodiments described herein, the dystrophin gene is edited with a pairof engineered meganucleases, resulting in the excision of exons 45-55and subsequent perfect ligation of the dystrophin gene. Removal of exons45-55 from the wild-type dystrophin gene can result in a dystrophinpolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 5.

As used herein, the term “perfect ligation” refers to the ligation(i.e., annealing) of all four bases of a 3′ overhang of a first cleavagesite with all four bases of a complementary 3′ overhang of a secondcleavage site in a dystrophin gene following cleavage by a pair ofengineered meganucleases of the invention. The recognition sequencestargeted by the disclosed engineered meganucleases have identical fourbasepair center sequences (e.g., GTAT), such that the first and secondcleavage sites will have complementary four basepair 3′ overhangs.Accordingly, each basepair of the first 3′ overhang pairs with itscomplement basepair on the second 3′ overhang, and ligation occursthrough a DNA ligase enzyme. Examples of sequences resulting from suchperfect ligations are set forth in SEQ ID NO: 32 (i.e., perfect ligationof the DMD 19-20 and DMD 35-36 recognition sequences) and SEQ ID NO: 34(i.e., perfect ligation of the DMD 19-20 and DMD 37-38 recognitionsequences).

As used herein, the term “Becker Muscular Dystrophy phenotype” refers toa less severe form of muscular dystrophy as compared to DMD. Individualshaving Becker Muscular Dystrophy still comprise mutations within thedystrophin gene, but express more functional dystrophin protein inmuscle cells (e.g., muscle precursor cells, skeletal muscle cells, andcardiac muscle cells) compared to individuals having DMD, generallyleading to a better clinical prognosis.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g., Cahill et al. (2006) Front. Biosci. 11:1958-76). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g., Cahill et al. (2006)). DNA repair by non-homologous end-joining iserror-prone and frequently results in the untemplated addition ordeletion of DNA sequences at the site of repair. In some instances,cleavage at a target recognition sequence results in NHEJ at a targetrecognition site. Nuclease-induced cleavage of a target site in thecoding sequence of a gene followed by DNA repair by non-homologous endjoining (NHEJ can introduce mutations into the coding sequence, such asframeshift mutations, that disrupt gene function. Thus, engineerednucleases can be used to effectively knock-out a gene in a population ofcells.

As used herein, the term “homology arms” or “sequences homologous tosequences flanking a nuclease cleavage site” refer to sequences flankingthe 5′ and 3′ ends of a nucleic acid molecule, which promote insertionof the nucleic acid molecule into a cleavage site generated by anuclease. In general, homology arms can have a length of at least 50base pairs, preferably at least 100 base pairs, and up to 2000 basepairs or more, and can have at least 90%, preferably at least 95%, ormore, sequence homology to their corresponding sequences in the genome.In some embodiments, the homology arms are about 500 base pairs.

As used herein, the term with respect to both amino acid sequences andnucleic acid sequences, the terms “percent identity,” “sequenceidentity,” “percentage similarity,” “sequence similarity” and the likerefer to a measure of the degree of similarity of two sequences basedupon an alignment of the sequences that maximizes similarity betweenaligned amino acid residues or nucleotides, and which is a function ofthe number of identical or similar residues or nucleotides, the numberof total residues or nucleotides, and the presence and length of gaps inthe sequence alignment. A variety of algorithms and computer programsare available for determining sequence similarity using standardparameters. As used herein, sequence similarity is measured using theBLASTp program for amino acid sequences and the BLASTn program fornucleic acid sequences, both of which are available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/), and aredescribed in, for example, Altschul et al. (1990) J. Mol. Biol.215:403-10; Gish & States (1993) Nature Genet. 3:266-72; Madden et al.(1996) Meth. Enzymol. 266:131-41; Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402; and Zhang et al. (2000) J. Comput. Biol. 7:203-14. Asused herein, percent similarity of two amino acid sequences is the scorebased upon the following parameters for the BLASTp algorithm: wordsize=3; gap opening penalty=−11; gap extension penalty=−1; and scoringmatrix=BLOSUM62. As used herein, percent similarity of two nucleic acidsequences is the score based upon the following parameters for theBLASTn algorithm: word size=11; gap opening penalty=−5; gap extensionpenalty=−2; match reward=1; and mismatch penalty=−3.

As used herein, the term “corresponding to” with respect tomodifications of two proteins or amino acid sequences is used toindicate that a specified modification in the first protein is asubstitution of the same amino acid residue as in the modification inthe second protein, and that the amino acid position of the modificationin the first protein corresponds to or aligns with the amino acidposition of the modification in the second protein when the two proteinsare subjected to standard sequence alignments (e.g., using the BLASTpprogram). Thus, the modification of residue “X” to amino acid “A” in thefirst protein will correspond to the modification of residue “Y” toamino acid “A” in the second protein if residues X and Y correspond toeach other in a sequence alignment and despite the fact that X and Y maybe different numbers.

As used herein, the term “recognition half-site,” “recognition sequencehalf-site,” or simply “half-site” means a nucleic acid sequence in adouble-stranded DNA molecule that is recognized and bound by a monomerof a homodimeric or heterodimeric meganuclease or by one subunit of asingle-chain meganuclease or by one subunit of a single-chainmeganuclease.

As used herein, the term “hypervariable region” refers to a localizedsequence within a meganuclease monomer or subunit that comprises aminoacids with relatively high variability. A hypervariable region cancomprise about 50-60 contiguous residues, about 53-57 contiguousresidues, or preferably about 56 residues. In some embodiments, theresidues of a hypervariable region may correspond to positions 24-79 orpositions 215-270 of any one of SEQ ID NOs: 36-59. A hypervariableregion can comprise one or more residues that contact DNA bases in arecognition sequence and can be modified to alter base preference of themonomer or subunit. A hypervariable region can also comprise one or moreresidues that bind to the DNA backbone when the meganuclease associateswith a double-stranded DNA recognition sequence. Such residues can bemodified to alter the binding affinity of the meganuclease for the DNAbackbone and the target recognition sequence. In different embodimentsof the invention, a hypervariable region may comprise between 1-20residues that exhibit variability and can be modified to influence basepreference and/or DNA-binding affinity. In particular embodiments, ahypervariable region comprises between about 15-20 residues that exhibitvariability and can be modified to influence base preference and/orDNA-binding affinity. In some embodiments, variable residues within ahypervariable region correspond to one or more of positions 24, 26, 28,30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ IDNOs: 36-59. In certain embodiments, variable residues within ahypervariable region can further correspond to residues 48, 50, and71-73 of any one of SEQ ID NOs: 36-59. In other embodiments, variableresidues within a hypervariable region correspond to one or more ofpositions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 239,241, 259, 261, 262, 263, 264, 266, and 268 of any one of SEQ ID NOs:36-59. In certain embodiments, variable residues within a hypervariableregion can further correspond to residues 239, 241, and 263-265 of anyone of SEQ TD NOs: 36-59.

The terms “increase” in the context of dystrophin protein or mRNA levelsrefers to any increase in the levels of dystrophin protein or mRNAexpression relative to a reference level including an increase ofdystrophin protein or mRNA expression of at least 1%, 2%, 3%, 4%, 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, ormore, when compared to a reference level or control. In someembodiments, an increase in dystrophin protein or mRNA levels refers toan increase in a shortened dystrophin polypeptide or mRNA transcript,for example, missing a portion of the polypeptide encoded by at leastone exon (e.g., a portion encoded by exons 45-55) or missing a portionof mRNA corresponding to exons 45-55 compared to the wild-typedystrophin polypeptide or gene.

As used herein, the term “reference level” in the context of dystrophinprotein or mRNA levels refers to a level of dystrophin protein or mRNAas measured in, for example, a control cell, control cell population ora control subject, at a previous time point in the control cell, thecontrol cell population or the subject undergoing treatment (e.g., apre-dose baseline level obtained from the control cell, control cellpopulation or subject), or a pre-defined threshold level of dystrophinprotein or mRNA (e.g., a threshold level identified through previousexperimentation).

As used herein, the term “a control” or “a control cell” refers to acell that provides a reference point for measuring changes in genotypeor phenotype of a genetically modified cell. A control cell maycomprise, for example: (a) a wild-type cell, i.e., of the same genotypeas the starting material for the genetic alteration which resulted inthe genetically modified cell; (b) a cell of the same genotype as thegenetically modified cell but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest); or, (c) a cell genetically identical to the geneticallymodified cell but which is not exposed to conditions or stimuli orfurther genetic modifications that would induce expression of alteredgenotype or phenotype. A control subject may comprise, for example: awild-type subject, i.e., of the same genotype as the starting subjectfor the genetic alteration which resulted in the genetically modifiedsubject (e.g., a subject having the same mutation in a dystrophin gene),which is not exposed to conditions or stimuli or further geneticmodifications that would induce expression of altered genotype orphenotype in the subject.

As used herein, the term “recombinant DNA construct,” “recombinantconstruct,” “expression cassette,” “expression construct,” “chimericconstruct,” “construct,” and “recombinant DNA fragment” are usedinterchangeably herein and are single or double-strandedpolynucleotides. A recombinant construct comprises an artificialcombination of nucleic acid fragments, including, without limitation,regulatory and coding sequences that are not found together in nature.For example, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource and arranged in a manner different than that found in nature.Such a construct may be used by itself or may be used in conjunctionwith a vector.

As used herein, the term “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used, then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant AAV vectors, or anyother vector known in the art suitable for delivering a gene to a targetcell. The skilled artisan is well aware of the genetic elements thatmust be present on the vector in order to successfully transform, selectand propagate host cells comprising any of the isolated nucleotides ornucleic acid sequences of the invention. In some embodiments, a “vector”also refers to a viral vector. Viral vectors can include, withoutlimitation, retroviral vectors, lentiviral vectors, adenoviral vectors,and AAV.

As used herein, the term “operably linked” is intended to mean afunctional linkage between two or more elements. For example, anoperable linkage between a nucleic acid sequence encoding a nuclease asdisclosed herein and a regulatory sequence (e.g., a promoter) is afunctional link that allows for expression of the nucleic acid sequenceencoding the nuclease. Operably linked elements may be contiguous ornon-contiguous. When used to refer to the joining of two protein codingregions, by operably linked is intended that the coding regions are inthe same reading frame.

As used herein, the terms “treatment” or “treating a subject” refers tothe administration of an engineered meganuclease described herein, or apolynucleotide encoding an engineered meganuclease described herein, ora pair of such engineered meganucleases or polynucleotides, to a subjecthaving DMD for the purpose of increasing levels of a dystrophin proteinin the subject. In some embodiments, expression of a shortened version(e.g., missing amino acids encoded by multiple exons) of the dystrophinprotein is increased. In some embodiments, expression of a version ofthe dystrophin protein, lacking the amino acids encoded by exons 45-55,is increased. Such treatment, in some embodiments, transitions the DMDphenotype to a Becker Dystrophy phenotype.

As used herein, the term “gc/kg” or “gene copies/kilogram” refers to thenumber of copies of a nucleic acid sequence encoding an engineeredmeganuclease described herein per weight in kilograms of a subject thatis administered a polynucleotide comprising the nucleic acid sequence.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to an amount sufficient to effect beneficial ordesirable biological and/or clinical results. The therapeuticallyeffective amount will vary depending on the formulation or compositionused, the disease and its severity and the age, weight, physicalcondition and responsiveness of the subject to be treated. In specificembodiments, an effective amount of an engineered meganuclease or pairof engineered meganucleases described herein, or polynucleotide or pairof polynucleotides encoding the same, or pharmaceutical compositionsdisclosed herein, increases the level of expression of a dystrophinprotein (e.g., a shortened dystrophin protein lacking the amino acidsencoded by exons 45-55) and ameliorates at least one symptom associatedwith DMD.

As used herein, the term “lipid nanoparticle” refers to a lipidcomposition having a typically spherical structure with an averagediameter between 10 and 1000 nm. In some formulations, lipidnanoparticles can comprise at least one cationic lipid, at least onenon-cationic lipid, and at least one conjugated lipid. Lipidnanoparticles known in the art that are suitable for encapsulatingnucleic acids, such as mRNA, are contemplated for use in the invention.

As used herein, the recitation of a numerical range for a variable isintended to convey that the present disclosure may be practiced with thevariable equal to any of the values within that range. Thus, for avariable which is inherently discrete, the variable can be equal to anyinteger value within the numerical range, including the end-points ofthe range. Similarly, for a variable that is inherently continuous, thevariable can be equal to any real value within the numerical range,including the end-points of the range. As an example, and withoutlimitation, a variable which is described as having values between 0 and2 can take the values 0, 1 or 2 if the variable is inherently discrete,and can take the values 0.0, 0.1, 0.01, 0.001, or any other realvalues≥0 and ≤2 if the variable is inherently continuous.

2.1 Principle of the Invention

The present disclosure is based, in part, on the hypothesis that certaindeletions in the dystrophin gene that give rise to the DMD phenotype canbe compensated for by utilizing pairs of endonucleases to strategicallydelete exons within the dystrophin gene in order to restore a normalreading frame within the gene. The DMD-Leiden Database indicates thatmost of the mutations that cause DMD are deletions of one or more wholeexons that cause a shift in reading frame. In many cases, the readingframe can be restored by eliminating the exon immediately before orafter the mutation. As shown in Table 3, 29 different Duchenne-causingmutations, representing ˜65% of patients, can be compensated for bydeleting a single exon adjacent to the mutation.

TABLE 3 Additional Frequency in Exon DMD-Leiden Exon(s) deleted inpatient to delete Database (%) 44, 44-47 43 5 35-43, 45, 45-54 44 818-44, 44, 46-47, 46-48, 45 13 46-49, 46-51, 46-53 45 46 7 51, 51-55 505 50, 45-50, 48-50, 49-50, 52, 51 15 52-63 51, 53, 53-55 52 3 45-52,48-52, 49-52, 50-52, 53 9 52

For example, a patient with disease due to the deletion of exon 45,which occurs in approximately 7% of patients, can be treated with atherapeutic that deletes exon 46. A therapeutic capable of deleting exon51 or exon 45 could be used to treat 15% and 13% of patients,respectively.

Notably, greater than 50% of all DMD-related mutations within thedystrophin gene are encompassed by exons 45 through 55. Thus, inparticular embodiments of the invention, exons 45 through 55 of thedystrophin gene will be removed in order to restore the normal readingframe of the gene. As disclosed herein, exon removal is achieved by theexpression of a pair of engineered meganucleases in muscle cells ormuscle precursor cells (e.g., a cardiac muscle cell or a skeletal musclecell) that generate a pair of cleavage sites in introns upstream of exon45 and downstream of exon 55, allowing for excision of the interveninggenomic region. Following this approach, a genetically modified cell(e.g., a muscle cell in a treated subject) will be able to make anamount of a shortened dystrophin protein from the Beckers phenotype,which is similar to micro-dystrophin approaches, without having toexpress a micro-dystrophin transgene. This shortened dystrophin may besufficient to rescue disease permanently, unlike other therapies thatrequire a multi-continuous treatment regimen.

Accordingly, it is envisioned that a single treatment will permanentlydelete exons from a percentage of cells in a subject. In someembodiments, these cells will be myoblasts (i.e., muscle cells) or othermuscle precursor cells that are capable of replicating and giving riseto whole muscle fibers that express functional (or semi-functional)dystrophin. If the frequency of exon deletion is low, however, it may benecessary to perform multiple treatments on each patient.

2.2 Meganucleases that Bind and Cleave Recognition Sequences Within aDystrophin Recognition Sequences

It is known in the art that it is possible to use a site-specificnuclease to make a DNA break in the genome of a living cell, and thatsuch a DNA break can result in permanent modification of the genome viamutagenic NHEJ repair or via homologous recombination with a transgenicDNA sequence. NHEJ can produce mutagenesis at the cleavage site,resulting in inactivation of the allele. NHEJ-associated mutagenesis mayinactivate an allele via generation of early stop codons, frameshiftmutations producing aberrant non-functional proteins, or could triggermechanisms such as nonsense-mediated mRNA decay. The use of nucleases toinduce mutagenesis via NHEJ can be used to target a specific mutation ora sequence present in a wild-type allele. Further, the use of nucleasesto induce a double-strand break in a target locus is known to stimulatehomologous recombination, particularly of transgenic DNA sequencesflanked by sequences that are homologous to the genomic target. In thismanner, exogenous polynucleotides can be inserted into a target locus.Such exogenous polynucleotides can encode any sequence or polypeptide ofinterest.

In particular embodiments, engineered meganucleases of the inventionhave been designed to bind and cleave a DMD 19-20 recognition sequence(SEQ ID NO: 6), a DMD 35-36 recognition sequence (SEQ ID NO: 10), or aDMD 37-38 recognition sequence (SEQ ID NO: 12). Exemplary meganucleasesthat bind and cleave the DMD 19-20 recognition sequence are provided inSEQ ID NOs: 36-44. Exemplary meganucleases that bind and cleave the DMD35-36 recognition sequence are provided in SEQ ID NOs: 45-52. Exemplarymeganucleases that bind and cleave the DMD 37-38 recognition sequenceare provided in SEQ ID NOs: 53-59. The sequence of each recognitionsequence, and the four base pair 3′ overhang produced when cleaved by anengineered meganuclease described herein, is provided in Table 4 below.

TABLE 4 Engineered Meganuclease Recognition Sequences 4 bp 3′Recognition Sequence SEQ ID NO: Overhang AAGGATTATGTATTACCTCCCG  6 GTATTAAGATTGGGTATGAGGGATAG  8 GTAT CTACATGGTGTATCTGACTAAG 10 GTATCTGGCCGAAGTATAGGAATATG 12 GTAT

In order to modify the dystrophin gene according to the presentdisclosure, a pair of engineered meganucleases described herein areutilized together in the same cell. Such pairs of engineeredmeganucleases were designed to generate a first cleavage site in anintron upstream of exon 45 and a second cleavage site in introndownstream of exon 55, allowing for removal of the intervening genomicsequence. Surprisingly, it was observed that excision of this genomicregion from the dystrophin gene, which is greater than 500,000 bp insize, could be accomplished with high efficiency. Moreover, themeganuclease recognition sequences were selected to have complementaryfour basepair 3′ overhangs following cleavage, and it was observed thatthe dystrophin gene could be repaired at high frequency by a perfectligation of the 3′ overhangs of the two cleavage sites. Such perfectlyligated recognition sequences contemplated herein are provided below inTable 5 below.

TABLE 5 Ligated Recognition Sequences SEQ ID Exon(s)Recognition Sequence Pair Ligated Recognition Sequence NO: RemovedDMD 19/20 and DMD 29/30 AAGGATTATGTATGAGGGATAG 30 45DMD 19/20 and DMD 35/36 AAGGATTATGTATCTGACTAAG 32 45-55DMD 19/20 and DMD 37/38 AAGGATTATGTATAGGAATATG 34 45-55

These recognition sequences are further selected to be within intronicsequences that are normally spliced out during post transcriptionalmodification cellular processes. This reduces the likelihood of amutation being introduced into the dystrophin gene and encodedpolypeptide.

Exemplary Engineered Meganucleases

Engineered meganucleases of the invention comprise a first subunit,comprising a HVR1 region, and a second subunit, comprising a HVR2region. Further, the first subunit binds to a first recognitionhalf-site in the recognition sequence (e.g., the DMD19 half-site), andthe second subunit binds to a second recognition half-site in therecognition sequence (e.g., the DMD20 half-site).

In particular embodiments, the meganucleases used to practice theinvention are single-chain meganucleases. A single-chain meganucleasecomprises an N-terminal subunit and a C-terminal subunit (i.e., thefirst and second subunits discussed above) joined by a linker peptide.Each of the two subunits recognizes and binds to a half-site of therecognition sequence and the site of DNA cleavage is at the middle ofthe recognition sequence near the interface of the two subunits. Asdiscussed, DNA strand breaks are offset by four base pairs such that DNAcleavage by a meganuclease generates a pair of four basepair 3′single-strand overhangs.

In embodiments where the engineered meganuclease is a single-chainmeganuclease, the first and second subunits can be oriented such thatthe first subunit, which comprises the HVR1 region and binds the firsthalf-site, is positioned as the N-terminal subunit, and the secondsubunit, which comprises the HVR2 region and binds the second half-site,is positioned as the C-terminal subunit. In alternative embodiments, thefirst and second subunits can be oriented such that the first subunit,which comprises the HVR1 region and binds the first half-site, ispositioned as the C-terminal subunit, and the second subunit, whichcomprises the HVR2 region and binds the second half-site, is positionedas the N-terminal subunit.

Exemplary DMD meganucleases of the invention are provided in SEQ ID NOs:36-59, and are summarized below in Tables 6-8.

TABLE 6 Exemplary engineered meganucleases that bind and cleave the DMD19-20 recognition sequence (SEQ ID NO: 6). AA DMD19 DMD19 *DMD19 DMD20DMD20 *DMD20 SEQ Subunit Subunit Subunit Subunit Subunit SubunitMeganuclease ID Residues SEQ ID % Residues SEQ ID % DMD 19-20x.13 367-153 84 100 198-344 108 100 DMD 19-20x.87 37 7-153 85 92.52 198-344 10995.24 DMD 19-20L.249 38 7-153 86 91.16 198-344 110 95.92 DMD 19-20L.30239 7-153 87 90.48 198-344 111 95.24 DMD 19-20L.329 40 7-153 88 91.16198-344 112 96.6 DMD 19-20L.374 41 7-153 89 91.84 198-344 113 95.92 DMD19-20L.375 42 7-153 90 91.84 198-344 114 95.92 DMD 19-20L.431 43 7-15391 91.16 198-344 115 96.60 DMD 19-20L.458 44 7-153 92 91.84 198-344 11696.60 “DMD19 Subunit %” and “DMD 20 Subunit %” represent the amino acidsequence identity between the DMD19-binding and DMD20-binding subunitregions of each meganuclease and the DMD19-binding and DMD20-bindingsubunit regions, respectively, of the DMD 19-20x.13 meganuclease.

TABLE 7 Exemplary engineered meganucleases that bind and cleave the DMD35-36 recognition sequence (SEQ ID NO: 10). AA DMD35 DMD35 *DMD35 DMD36DMD36 *DMD36 SEQ Subunit Subunit Subunit Subunit Subunit SubunitMeganuclease ID Residues SEQ ID % Residues SEQ ID % DMD 35-36x.63 457-153 93 100 198-344 117 100 DMD 35-36x.81 46 7-153 94 97.96 198-344 11893.88 DMD 35-36L.195 47 7-153 95 100 198-344 119 93.20 DMD 35-36L.282 487-153 96 99.32 198-344 120 93.20 DMD 35-36L.349 49 7-153 97 100 198-344121 93.20 DMD 35-36L.376 50 7-153 98 100 198-344 122 93.20 DMD35-36L.457 51 7-153 99 99.32 198-344 123 92.52 DMD 35-36L.469 52 7-153100 98.64 198-344 124 95.52 “DMD35 Subunit %” and “DMD36 Subunit %”represent the amino acid sequence identity between the DMD35-binding andDMD36-binding subunit regions of each meganuclease and the DMD35-bindingand DMD36-binding subunit regions, respectively, of the DMD 35-36x.63meganuclease.

TABLE 8 Exemplary engineered meganucleases that bind and cleave the DMD37-38 recognition sequence (SEQ ID NO: 12). AA DMD37 DMD37 *DMD37 DMD38DMD38 *DMD38 SEQ Subunit Subunit Subunit Subunit Subunit SubunitMeganuclease ID Residues SEQ ID % Residues SEQ ID % DMD 37-38x.15 537-153 101 100 198-344 125 100 DMD 37-38x.66 54 7-153 102 98.64 198-344126 96.60 DMD 37-38x.79 55 7-153 103 99.32 198-344 127 95.24 DMD37-38.L166 56 7-153 104 91.84 198-344 128 94.56 DMD 37-38L.478 57 7-153105 91.84 198-344 129 93.88 DMD 37-38L.512 58 7-153 106 91.84 198-344130 94.56 DMD 37-38L.528 59 7-153 107 90.48 198-344 131 94.56 “DMD37Subunit %” and “DMD38 Subunit %” represent the amino acid sequenceidentity between the DMD37-binding and DMD38-binding subunit regions ofeach meganuclease and the DMD37-binding and DMD38-binding subunitregions, respectively, of the DMD 37-38x.15 meganuclease.

In certain embodiments of the invention, the engineered meganucleasebinds and cleaves a recognition sequence comprising SEQ ID NO: 6 (i.e.,the DMD 19-20 recognition sequence) within a dystrophin gene, whereinthe engineered meganuclease comprises a first subunit and a secondsubunit, wherein the first subunit binds to a first recognitionhalf-site of the recognition sequence and comprises a HVR1 region, andwherein the second subunit binds to a second recognition half-site ofthe recognition sequence and comprises a HVR2 region. Exemplary DMD19-20 meganucleases are described below.

DMD 19-20x.13 (SEQ ID NO: 36)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 36. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 36. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 36. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 36. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:36 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 36.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 36. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 36. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 36. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 36. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 36with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 36.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 36. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 36. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 36. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 36. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 241 of SEQ ID NO: 36. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 263 of SEQ ID NO: 36. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 36. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 36 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 36.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 36. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 36. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 36. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 36. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 36. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 36 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 36.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 36. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 36. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 50. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 50.

DMD 19-20x.87 (SEQ ID NO: 37)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 37. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 37. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 37. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 37. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:37 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 37.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 37. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 37. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 37. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 37. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 37with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 37.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 37. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 37. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 37. In some embodiments,the HVR2 region comprises Y, R. K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 37. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 37. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 37. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 37. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 37 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 37.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 37. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 37. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 37. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 37. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 37 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 37.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 37. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 37. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 51. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 51.

DMD 19-20L.249 (SEQ ID NO: 38)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 38. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 38. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 38. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 38. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:38 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 38.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ TD NO: 38. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 38. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 38. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 38. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 38. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 38 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 38.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 38. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 38. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 38. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 38. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 38. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 38 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 38.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 38. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 38. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 38. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 38. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 38 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 38.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 38. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 38. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 52. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 52.

DMD 19-20L.302 (SEQ ID NO: 39)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 39. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 39. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 39. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 39. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:39 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 39.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ TD NO: 39. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 39. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 39. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 39. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 39. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 39 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ TD NO: 39.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 39. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 39. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 39. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 39. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 236 of SEQ ID NO: 39. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 39. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 39 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 39.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 39. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 39. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 39. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 39. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 39 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 39.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 39. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 39. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NOs: 53. In some embodiments, the engineeredmeganuclease is encoded by a nucleic acid sequence set forth in SEQ IDNO: 53.

DMD 19-20L.329 (SEQ ID NO: 40)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 40. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 40. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 40. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 40. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:40 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 40.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 40. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 40. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 40. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 40. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 40with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 40.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 40. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 40. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 40. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 40. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 40. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 40 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 40.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 40. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 40. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 40. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 40. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 40. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 40 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 40.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 40. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 40. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 54. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 54.

DMD 19-20L.374 (SEQ ID NO: 41)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 41. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 41. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 41. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 40. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:41 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 41.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 41. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 41. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 41. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 41. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 41with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 41.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 41. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 41. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 41. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ TD NO: 41. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 41. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 41 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 41.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 41. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 41. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 41. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 41. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 41 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 41.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 41. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 41. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 65. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 65.

DMD 19-20L.375 (SEQ ID NO: 42)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 42. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 42. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 42. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 42. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:42 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 42.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 42. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 42. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 42. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 42. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 42with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 42.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 42. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 42. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 42. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 42. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 42. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 42 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 42.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 42. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 42. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 42. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ TD NO: 42. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 42 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 42.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 42. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 42. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 66. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 66.

DMD 19-20L.431 (SEQ ID NO: 43)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 43. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 43. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 43. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 43. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:43 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 43.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 43. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 43. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 43. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 43. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 43. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 43 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 43.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 43. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 43. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 43. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 43. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 43. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 43 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 43.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 43. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 43. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 43. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 43. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 43. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 43 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 43.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 43. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 43. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 67. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 67.

DMD 19-20L.458 (SEQ ID NO: 44)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 44. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 44. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 44. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 44. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 44.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 44. Insome embodiments, the first subunit comprises G, S. or A at a residuecorresponding to residue 19 of SEQ ID NO: 44. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 44. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 44. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 44with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 44.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 44. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 44. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 44. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 44. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 44. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 44 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 44.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 44. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 44. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 44. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 44. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 44. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 44 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 44.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 44. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 44. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 68. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 68.

In certain embodiments of the invention, the engineered meganucleasebinds and cleaves a recognition sequence comprising SEQ ID NO: 10 (i.e.,the DMD 35-36 recognition sequence) within a dystrophin gene, whereinthe engineered meganuclease comprises a first subunit and a secondsubunit, wherein the first subunit hinds to a first recognitionhalf-site of the recognition sequence and comprises a firsthypervariable (HVR1) region, and wherein the second subunit binds to asecond recognition half-site of the recognition sequence and comprises asecond hypervariable (HVR2) region. Exemplary DMD 35-36 meganucleasesare described below.

DMD 35-36x.63 (SEQ ID NO: 45)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 45. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 45. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 45. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 45. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:45 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 45.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 45. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 45. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 45. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 45. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 45. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 45 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 45.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 45. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 45. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 45. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 45. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 45. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 45. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 250 of SEQ ID NO: 45. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 263 of SEQ ID NO: 45. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 45. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 45 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 45. In some embodiments, the second subunitcomprises an amino acid sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toresidues 198-344 of SEQ ID NO: 45. In some embodiments, the secondsubunit comprises G, S, or A at a residue corresponding to residue 210of SEQ ID NO: 45. In some embodiments, the second subunit comprises E,Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 45. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 45. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 45 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 45.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 45. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 45. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 69. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 69.

DMD 35-36x.81 (SEQ ID NO: 46)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 46. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 46. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 46. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 46. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:46 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 46.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 46. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 46. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 46. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 46. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 46. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 46 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 46.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 46. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 46. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 46. In some embodiments,the HVR2 region comprises Y, R. K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 46. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 46. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 46. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of SEQ ID NO: 46. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 46. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 46 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 46.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 46. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 46. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 46. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 46. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 46 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 46.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 46. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 46. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 70. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 70.

DMD 35-36L.195 (SEQ ID NO: 47)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 47. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 47. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 47. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 47. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:47 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 47.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 47. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 47. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 47. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 47. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 47. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 47 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 47.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 47. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 47. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 47. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 47. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 47. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 47. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 47. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 47 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 47.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 47. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 47. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 47. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 47. Insome embodiments, the second subunit comprises residues 198-344 of SEQTD NO: 47 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 47.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 47. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 47. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 71. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 71.

DMD 35-36L.282 (SEQ ID NO: 48)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 48. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 48. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 48. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 48. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:48 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 48.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 48. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 48. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 48. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 48. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 48. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 48 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 48.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 48. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 48. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 48. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 48. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 48. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 48. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ TD NO: 48. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 48 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 48.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 48. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 48. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 48. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 48. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 48 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 48.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 48. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 48. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 72. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 72.

DMD 35-36L.349 (SEQ ID NO: 49)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 49. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 49. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 49. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 49. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:49 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 49.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 49. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 49. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 49. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 49. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 49. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 49 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 49.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 49. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 49. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 49. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 49. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 49. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 49. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 49. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 49 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 49.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 49. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 49. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 49. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 49. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 49 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 49.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 49. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 49. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 73. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 73.

DMD 35-36L.376 (SEQ ID NO: 50)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 50. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 50. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 50. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 50. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:50 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 50.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 50. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 50. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 50. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 50. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 50. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 50 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 50.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 50. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 50. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 50. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 50. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 50. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 50. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 50. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 50 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 50.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 50. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 50. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 50. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 50. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 50 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 50.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 50. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 50. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 74. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 74.

DMD 35-36L.457 (SEQ ID NO: 51)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 51. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 51. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 51. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 51. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:51 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 51.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 51. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 51. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 51. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 51. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 51. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 51 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 51.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 51. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 51. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 51. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 51. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 51. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 51. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 51. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 51 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 51.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 51. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 51. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 51. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 51. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 51 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 51.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 51. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 51. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 75. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 75.

DMD 35-36L.469 (SEQ ID NO: 52)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 52. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 52. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 52. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 52. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 52.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 52. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 52. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 52. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 52. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 52with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 52.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 52. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 52. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 52. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 52. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 52. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 52. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 52. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 52.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 52. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 52. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 52. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 52. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 52. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 52 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 52.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 52. In some embodiments, the engineeredmeganuclease comprises an amino acid sequence of SEQ ID NO: 52. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 76. In some embodiments, the engineered meganucleaseis encoded by a nucleic acid sequence set forth in SEQ ID NO: 76.

In certain embodiments of the invention, the engineered meganucleasebinds and cleaves a recognition sequence comprising SEQ ID NO: 12 (i.e.,the DMD 37-38 recognition sequence) within a dystrophin gene, whereinthe engineered meganuclease comprises a first subunit and a secondsubunit, wherein the first subunit binds to a first recognitionhalf-site of the recognition sequence and comprises a firsthypervariable (HVR1) region, and wherein the second subunit binds to asecond recognition half-site of the recognition sequence and comprises asecond hypervariable (HVR2) region. Exemplary DMD 37-38 meganucleasesare described below.

DMD 37-38x.15 (SEQ ID NO: 53)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 53. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 53. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 53. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 53. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:53 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 53.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 53. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 53. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 53. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 53. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 53. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 53 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ TD NO: 53.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 53. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 53. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 53. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 53. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 53. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 53. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 264 of SEQ ID NO: 53. Insome embodiments, the HVR2 region comprises residues 215-270 of SEQ IDNO: 53 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR2 region comprises residues215-270 of SEQ ID NO: 53.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 53. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 53. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 53. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 53. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 53 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 53.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 53. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 53. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 77. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 77.

DMD 37-38x.66 (SEQ ID NO: 54)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 54. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 54. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 54. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 54. Insome embodiments, the HVR1 region comprises a residue corresponding toresidue 64 of SEQ ID NO: 54. In some embodiments, the HVR1 regioncomprises residues 24-79 of SEQ ID NO: 54 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR1 region comprises residues 24-79 of SEQ ID NO: 54.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 54. Insome embodiments, the first subunit comprises G, S. or A at a residuecorresponding to residue 19 of SEQ ID NO: 54. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 54. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 54. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 54. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 54 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 54.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 54. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 54. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 54. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 54. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 241 of SEQ ID NO: 54. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 54. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 54 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 54.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 54. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 54. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 54. In some embodiments, the second subunitcomprises a residue corresponding to residue 330 of SEQ ID NO: 54. Insome embodiments, the second subunit comprises residues 198-344 of SEQID NO: 54 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the second subunit comprisesresidues 198-344 of SEQ ID NO: 54.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 54. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 54. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 78. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 78.

DMD 37-38x.79 (SEQ ID NO: 55)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 55. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 55. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 55. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 55. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:55 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 55.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 55. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 55. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 55. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 55. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 55. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 55 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 55.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 55. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 55. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 55. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 55. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 239 of SEQ ID NO: 55. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 241 of SEQ ID NO: 55. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 255 of SEQ ID NO: 55. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 55. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 55 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 55.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 55. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 55. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 55. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 55. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 55. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 55 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 55.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 55. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 55. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 79. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 79.

DMD 37-38L.166 (SEQ ID NO: 56)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 56. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 56. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 56. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 56. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:56 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 56.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 56. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 56. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 56. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 56. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 56with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ TD NO: 56.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 56. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 56. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 56. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 56. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of SEQ ID NO: 56. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 56. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 56 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 56.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 56. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 56. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 56. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 56. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 56. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 56 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 56.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 56. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 56. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 80. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 80.

DMD 37-38L.478 (SEQ ID NO: 57)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 57. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 57. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 57. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 57. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:57 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 57.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 57. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 57. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 57. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 57. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 57. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 57 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 57.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 57. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 57. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 57. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 57. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of SEQ ID NO: 57. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 57. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 57 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 57.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 57. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 57. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 57. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 57. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 57. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 57 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 57.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 57. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ TD NO: 57. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 81. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 81.

DMD 37-38L.512 (SEQ ID NO: 58)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 58. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 58. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 58. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 58. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:58 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 58.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 58. Insome embodiments, the first subunit comprises G, S, or A at a residuecorresponding to residue 19 of SEQ ID NO: 58. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 58. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 58. In someembodiments, the first subunit comprises a residue corresponding toresidue 80 of SEQ ID NO: 58. In some embodiments, the first subunitcomprises residues 7-153 of SEQ ID NO: 58 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thefirst subunit comprises residues 7-153 of SEQ ID NO: 58.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 58. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 58. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 58. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 58. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of SEQ ID NO: 58. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 58. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 58 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 58.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 58. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 58. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 58. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 58. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 58. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 58 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 58.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 58. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 58. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 82. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 82.

DMD 37-38L.528 (SEQ ID NO: 59)

In some embodiments, the HVR1 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 24-79 of SEQ ID NO: 59. In some embodiments, the HVR1 regioncomprises one or more residues corresponding to residues 24, 26, 28, 30,32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 59. In someembodiments, the HVR1 region comprises residues corresponding toresidues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77of SEQ ID NO: 59. In some embodiments, the HVR1 region comprises Y, R,K, or D at a residue corresponding to residue 66 of SEQ ID NO: 59. Insome embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO:59 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidsubstitutions. In some embodiments, the HVR1 region comprises residues24-79 of SEQ ID NO: 59.

In some embodiments, the first subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 7-153 of SEQ ID NO: 59. Insome embodiments, the first subunit comprises G, S. or A at a residuecorresponding to residue 19 of SEQ ID NO: 59. In some embodiments, thefirst subunit comprises a residue corresponding to residue 19 of SEQ IDNO: 59. In some embodiments, the first subunit comprises E, Q, or K at aresidue corresponding to residue 80 of SEQ ID NO: 59. In someembodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 59with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidsubstitutions. In some embodiments, the first subunit comprises residues7-153 of SEQ ID NO: 59.

In some embodiments, the HVR2 region comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to an amino acid sequence corresponding toresidues 215-270 of SEQ ID NO: 59. In some embodiments, the HVR2 regioncomprises one or more residues corresponding to residues 215, 217, 219,221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ IDNO: 59. In some embodiments, the HVR2 region comprises residuescorresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233,235, 237, 259, 261, 266, and 268 of SEQ ID NO: 59. In some embodiments,the HVR2 region comprises Y, R, K, or D at a residue corresponding toresidue 257 of SEQ ID NO: 59. In some embodiments, the HVR2 regioncomprises a residue corresponding to residue 263 of SEQ ID NO: 59. Insome embodiments, the HVR2 region comprises a residue corresponding toresidue 264 of SEQ ID NO: 59. In some embodiments, the HVR2 regioncomprises residues 215-270 of SEQ ID NO: 59 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, theHVR2 region comprises residues 215-270 of SEQ ID NO: 59.

In some embodiments, the second subunit comprises an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to residues 198-344 of SEQ ID NO: 59. Insome embodiments, the second subunit comprises G, S, or A at a residuecorresponding to residue 210 of SEQ ID NO: 59. In some embodiments, thesecond subunit comprises E, Q, or K at a residue corresponding toresidue 271 of SEQ ID NO: 59. In some embodiments, the second subunitcomprises a residue corresponding to residue 271 of SEQ ID NO: 59. Insome embodiments, the second subunit comprises a residue correspondingto residue 330 of SEQ ID NO: 59. In some embodiments, the second subunitcomprises residues 198-344 of SEQ ID NO: 59 with up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, thesecond subunit comprises residues 198-344 of SEQ ID NO: 59.

In some embodiments, the engineered meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinssaid first subunit and said second subunit. In some embodiments, theengineered meganuclease comprises an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity SEQ ID NO: 59. In some embodiments, the engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO: 59. In someembodiments, the engineered meganuclease is encoded by a nucleicsequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to a nucleic acid sequence setforth in SEQ ID NO: 83. In some embodiments, the engineered meganucleaseis encoded by a nucleic sequence set forth in SEQ ID NO: 83.

2.3 Methods for Delivering and Expressing Engineered Meganucleases

In different aspects, the invention provides engineered meganucleasesdescribed herein that are useful for binding and cleaving recognitionsequences within a dystrophin gene of a cell (e.g., the human dystrophingene). The invention provides various methods for modifying a dystrophingene in cells using engineered meganucleases described herein, methodsfor making genetically modified cells comprising a modified dystrophingene, and methods of modifying a dystrophin gene in a target cell in asubject. In further aspects, the invention provides methods for treatingDMD in a subject by administering the engineered meganucleases describedherein, or polynucleotides encoding the same, to a subject, in somecases as part of a pharmaceutical composition.

In each case, it is envisioned that the engineered meganucleases, orpolynucleotides encoding the same, are introduced into cells, such asmuscle cells or muscle precursor cells capable of expressing adystrophin protein. Engineered meganucleases described herein can bedelivered into a cell in the form of protein or, preferably, as apolynucleotide encoding the engineered meganuclease. Suchpolynucleotides can be, for example, DNA (e.g., circular or linearizedplasmid DNA, PCR products, or a viral genome) or RNA (e.g., mRNA).

Detection and Expression

Expression of a modified dystrophin (i.e., a gene lacking exons 45-55,or a protein lacking amino acids encoded by exons 45-55) in agenetically modified cell or subject can be detected using standardmethods in the art. For example, levels of such modified dystrophin maybe assessed based on the level of any variable associated withdystrophin gene expression, e.g., dystrophin mRNA levels or dystrophinprotein levels. Increased levels or expression of such modifieddystrophin may be assessed by an increase in an absolute or relativelevel of one or more of these variables compared with a reference level.Such modified dystrophin levels may be measured in a biological sampleisolated from a subject, such as a tissue biopsy or a bodily fluidincluding blood, serum, plasma, cerebrospinal fluid, or urine.Optionally, such modified dystrophin levels are normalized to a standardprotein or substance in the sample. Further, such modified dystrophinlevels can be assessed any time before, during, or after treatment inaccordance with the methods herein.

In various aspects, the methods described herein can increase proteinlevels of a modified dystrophin (i.e., lacking amino acids encoded byexons 45-55) in a genetically modified cell, target cell, or subject(e.g., as measured in a cell, a tissue, an organ, or a biological sampleobtained from the subject), to at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more, of areference level (i.e., protein level of dystrophin in a wild-type cellor subject). In some embodiments, the methods herein are effective toincrease the level of such modified dystrophin protein to about 10% toabout 100% (e.g., 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%,70%-80%, 80%-90%, 90%-100%, or more) of a reference level of dystrophin(i.e., protein level of dystrophin in a wild-type cell or subject).

Introduction of Engineered Meganucleases into Cells

Engineered meganuclease proteins disclosed herein, or polynucleotidesencoding the same, can be delivered into cells to cleave genomic DNA bya variety of different mechanisms known in the art, including thosefurther detailed herein below.

Engineered meganucleases disclosed herein can be delivered into a cellin the form of protein or, preferably, as a polynucleotide comprising anucleic acid sequence encoding the engineered meganuclease. Suchpolynucleotides can be, for example, DNA (e.g., circular or linearizedplasmid DNA, PCR products, or viral genomes) or RNA (e.g., mRNA).

For embodiments in which the engineered meganuclease coding sequence isdelivered in DNA form, it should be operably linked to a promoter tofacilitate transcription of the meganuclease gene. Mammalian promoterssuitable for the invention include constitutive promoters such as thecytomegalovirus early (CMV) promoter (Thomsen et al. (1984) Proc NatlAcad Sci USA. 81:659-63) or the SV40 early promoter (Benoist & Chambon(1981) Nature 290:304-10) as well as inducible promoters such as thetetracycline-inducible promoter (Dingermann et al. (1992) Mol Cell Biol.12:4038-45). An engineered meganuclease of the invention can also beoperably linked to a synthetic promoter. Synthetic promoters caninclude, without limitation, the JeT promoter (WO 2002/012514).

In specific embodiments, a nucleic acid sequence encoding an engineerednuclease of the invention is operably linked to a tissue-specificpromoter, such as a muscle-specific promoter. In some particularembodiments, the promoter is capable of expressing an engineeredmeganuclease described herein in a muscle precursor cell (e.g.,satellite cell or stem cell). Exemplary and non-limiting musclepromoters include C5-12 (Liu et al. (2004) Hum Gene Ther. 15:783-92),the muscle-specific creatine kinase (MCK) promoter (Yuasa et al. (2002)Gene Ther. 9:1576-88), or the smooth muscle 22 (SM22) promoter (Haase etal. (2013) BMC Biotechnol. 13:49-54). In some embodiments, themuscle-specific promoter comprises the sequence according to any one ofSEQ ID NOs: 169-181. In some embodiments, the muscle-specific promotercomprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a C5-12 promoter comprising SEQ ID NO: 169. In someembodiments, the muscle-specific promoter comprises a sequence 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a murine MCKpromoter comprising SEQ ID NO: 170. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a human MCK promotercomprising SEQ ID NO: 171. In some embodiments, the muscle-specificpromoter comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to a MCK enhancer comprising SEQ ID NO: 172.In some embodiments, the muscle-specific promoter comprises a sequence90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amodified MCK enhancer comprising SEQ ID NO: 173. In some embodiments,the muscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a spc 5-12 promotercomprising SEQ ID NO: 174. In some embodiments, the muscle-specificpromoter comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to a MHCK7 promoter comprising SEQ ID NO:175. In some embodiments, the muscle-specific promoter comprises asequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to a CK8 promoter comprising SEQ ID NO: 176. In someembodiments, the muscle-specific promoter comprises a sequence 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a SK-CRM4promoter comprising SEQ ID NO: 177. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a SP-301 promotercomprising SEQ ID NO: 178. In some embodiments, the muscle-specificpromoter comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to a SP-817 promoter comprising SEQ ID NO:179. In some embodiments, the muscle-specific promoter comprises asequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to a SP-905 promoter comprising SEQ ID NO: 180. In someembodiments, the muscle-specific promoter comprises a sequence 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a musclehybrid promoter comprising SEQ ID NO: 181.

In some embodiments, wherein a single polynucleotide comprises twoseparate nucleic acid sequences each encoding an engineered meganucleasedescribed herein, the meganuclease genes are operably linked to twoseparate promoters. In alternative embodiments, the two meganucleasegenes are operably linked to a single promoter, and in some examples canbe separated by an internal-ribosome entry site (IRES) or a 2A peptidesequence (Szymezak & Vignali (2005) Expert Opin Biol Ther. 5:627-38).Such 2A peptide sequences can include, for example, a T2A, P2A, E2A, orF2A sequence.

In specific embodiments, a polynucleotide comprising a nucleic acidsequence encoding an engineered meganuclease described herein isdelivered on a recombinant DNA construct or expression cassette. Forexample, the recombinant DNA construct can comprise an expressioncassette (i.e., “cassette”) comprising a promoter and a nucleic acidsequence encoding an engineered meganuclease described herein.

In another particular embodiment, a polynucleotide comprising a nucleicacid sequence encoding an engineered meganuclease described herein isintroduced into the cell using a single-stranded DNA template. Thesingle-stranded DNA can further comprise a 5′ and/or a 3′ AAV invertedterminal repeat (ITR) upstream and/or downstream of the sequenceencoding the engineered nuclease. The single-stranded DNA can furthercomprise a 5′ and/or a 3′ homology arm upstream and/or downstream of thesequence encoding the engineered meganuclease.

In another particular embodiment, a polynucleotide comprising a nucleicacid sequence encoding an engineered meganuclease described herein canbe introduced into a cell using a linearized DNA template. Suchlinearized DNA templates can be produced by methods known in the art.For example, a plasmid DNA encoding a nuclease can be digested by one ormore restriction enzymes such that the circular plasmid DNA islinearized prior to being introduced into a cell.

In some embodiments, mRNA encoding an engineered meganuclease describedherein is delivered to a cell because this reduces the likelihood thatthe gene encoding the engineered meganuclease will integrate into thegenome of the cell. Such mRNA can be produced using methods known in theart such as in vitro transcription. In some embodiments, the mRNA is 5′capped using 7-methyl-guanosine, anti-reverse cap analogs (ARCA) (U.S.Pat. No. 7,074,596), CleanCap® analogs such as Cap 1 analogs (Trilink,San Diego, CA), or enzymatically capped using vaccinia capping enzyme orsimilar. In some embodiments, the mRNA may be polyadenylated. The mRNAmay contain various 5′ and 3′ untranslated sequence elements to enhanceexpression the encoded engineered meganuclease and/or stability of themRNA itself. Such elements can include, for example, posttranslationalregulatory elements such as a woodchuck hepatitis virusposttranslational regulatory element. The mRNA may contain nucleosideanalogs or naturally-occurring nucleosides, such as pseudouridine,5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thiouridine.Additional nucleoside analogs include, for example, those described inU.S. Pat. No. 8,278,036.

In some embodiments, the meganuclease proteins, or DNA/mRNA encoding themeganuclease, are coupled to a cell penetrating peptide or targetingligand to facilitate cellular uptake. Examples of cell penetratingpeptides known in the art include poly-arginine (Jearawiriyapaisarn etal. (2008) Mol Ther. 16:1624-29), TAT peptide from the HIV virus (Hudeczet al. (2005) Med. Res. Rev. 25:679-736), MPG (Simeoni et al. (2003)Nucleic Acids Res. 31:2717-24), Pep-1 (Deshayes et al. (2004)Biochemistry 43:7698-7706, and HSV-1 VP-22 (Deshayes et al. (2005) CellMol Life Sci. 62:1839-49). In an alternative embodiment, engineerednucleases, or DNA/mRNA encoding nucleases, are coupled covalently ornon-covalently to an antibody that recognizes a specific cell-surfacereceptor expressed on target cells such that the nucleaseprotein/DNA/mRNA binds to and is internalized by the target cells.Alternatively, engineered nuclease protein/DNA/mRNA can be coupledcovalently or non-covalently to the natural ligand (or a portion of thenatural ligand) for such a cell-surface receptor. (McCall et al. (2014)Tissue Barriers. 2(4):e944449; Dinda et al. (2013) Curr. Pharm.Biotechnol. 14:1264-74; Kang et al. (2014) Curr. Pharm. Biotechnol.15:220-30; and Qian et al. (2014) Expert Opin. Drug Metab Toxicol.10:1491-508).

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are encapsulated within biodegradable hydrogels forinjection or implantation within the desired region of the liver (e.g.,in proximity to hepatic sinusoidal endothelial cells or hematopoieticendothelial cells, or progenitor cells which differentiate into thesame). Hydrogels can provide sustained and tunable release of thetherapeutic payload to the desired region of the target tissue withoutthe need for frequent injections, and stimuli-responsive materials(e.g., temperature- and pH-responsive hydrogels) can be designed torelease the payload in response to environmental or externally appliedcues (Derwent et al. (2008) Trans Am. Ophthalmol. Soc. 106:206-14).

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are coupled covalently or, preferably, non-covalently toa nanoparticle or encapsulated within such a nanoparticle using methodsknown in the art (Sharma et al. (2014) Biomed. Res. Int. 2014:156010). Ananoparticle is a nanoscale delivery system whose length scale is <1 μm,preferably <100 nm. Such nanoparticles may be designed using a corecomposed of metal, lipid, polymer, or biological macromolecule, andmultiple copies of the meganuclease proteins, mRNA, or DNA can beattached to or encapsulated with the nanoparticle core. This increasesthe copy number of the protein/mRNA/DNA that is delivered to each celland, so, increases the intracellular expression of each meganuclease tomaximize the likelihood that the target recognition sequences will becut. The surface of such nanoparticles may be further modified withpolymers or lipids (e.g., chitosan, cationic polymers, or cationiclipids) to form a core-shell nanoparticle whose surface confersadditional functionalities to enhance cellular delivery and uptake ofthe payload (Jian et al. (2012) Biomaterials. 33:7621-30). Nanoparticlesmay additionally be advantageously coupled to targeting molecules todirect the nanoparticle to the appropriate cell type and/or increase thelikelihood of cellular uptake. Examples of such targeting moleculesinclude antibodies specific for cell-surface receptors and the naturalligands (or portions of the natural ligands) for cell surface receptors.

In some embodiments, the meganuclease proteins, or DNA/mRNA encodingmeganucleases, are encapsulated within liposomes or complexed usingcationic lipids (see, e.g., LIPOFECTAMINE™, Life Technologies Corp.,Carlsbad, CA; Zuris et al. (2015) Nat. Biotechnol. 33:73-80; Mishra etal. (2011) J. Drug Deliv. 2011:863734). The liposome and lipoplexformulations can protect the payload from degradation, enhanceaccumulation and retention at the target site, and facilitate cellularuptake and delivery efficiency through fusion with and/or disruption ofthe cellular membranes of the target cells.

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are encapsulated within polymeric scaffolds (e.g., PLGA)or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al.(2011) Ther Deliv. 2:523-36). Polymeric carriers can be designed toprovide tunable drug release rates through control of polymer erosionand drug diffusion, and high drug encapsulation efficiencies can offerprotection of the therapeutic payload until intracellular delivery tothe desired target cell population.

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are combined with amphiphilic molecules thatself-assemble into micelles (Tong et al. (2007) J. Gene Med. 9:956-66).Polymeric micelles may include a micellar shell formed with ahydrophilic polymer (e.g., polyethyleneglycol) that can preventaggregation, mask charge interactions, and reduce nonspecificinteractions.

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are formulated into an emulsion or a nanoemulsion (i.e.,having an average particle diameter of <1 nm) for administration and/ordelivery to the target cell. The term “emulsion” refers to, withoutlimitation, any oil-in-water, water-in-oil, water-in-oil-in-water, oroil-in-water-in-oil dispersions or droplets, including lipid structuresthat can form as a result of hydrophobic forces that drive apolarresidues (e.g., long hydrocarbon chains) away from water and polar headgroups toward water, when a water immiscible phase is mixed with anaqueous phase. These other lipid structures include, but are not limitedto, unilamellar, paucilamellar, and multilamellar lipid vesicles,micelles, and lamellar phases. Emulsions are composed of an aqueousphase and a lipophilic phase (typically containing an oil and an organicsolvent). Emulsions also frequently contain one or more surfactants.Nanoemulsion formulations are well known, for example, as described inU.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, 6,559,189, and7,767,216, each of which is incorporated herein by reference in itsentirety.

In some embodiments, meganuclease proteins, or DNA/mRNA encodingmeganucleases, are covalently attached to, or non-covalently associatedwith, multifunctional polymer conjugates, DNA dendrimers, and polymericdendrimers (Mastorakos et al. (2015) Nanoscale. 7:3845-56; Cheng et al.(2008) J. Pharm Sci. 97:123-43). The dendrimer generation can controlthe payload capacity and size and can provide a high payload capacity.Moreover, display of multiple surface groups can be leveraged to improvestability, reduce nonspecific interactions, and enhance cell-specifictargeting and drug release.

In some embodiments, polynucleotides comprising a nucleic acid sequenceencoding an engineered meganuclease described herein are introduced intoa cell using a recombinant virus (i.e., a recombinant viral vector).Such recombinant viruses are known in the art and include recombinantretroviruses, recombinant lentiviruses, recombinant adenoviruses, andrecombinant AAVs (reviewed in Vannucci et al. (2013) New Microbiol.36:1-22). Recombinant AAVs useful in the invention can have any serotypethat allows for transduction of the virus into a target cell type andexpression of the meganuclease gene in the target cell. For example, insome embodiments, recombinant AAVs have a serotype (i.e., a capsid) ofAAV1, AAV2, AAV5 AAV6, AAV7, AAV8, AAV9, AAV12, or AAVrh.74. It is knownin the art that different AAVs tend to localize to different tissues(Wang et al. (2014) Expert Opin Drug Deliv 11:345-34.). The AAVrh.74serotype, which is closely related to AAV8, has further been describedas targeting muscle tissue including skeletal muscle and cardiac muscletissue (Mendell et al. (2020) JAMA Neurol. 77:1122-31). Accordingly, insome embodiments, the AAV serotype is AAV1. In some embodiments, the AAVserotype is AAV2. In some embodiments, the AAV serotype is AAV5. In someembodiments, the AAV serotype is AAV6. In some embodiments, the AAVserotype is AAV7. In some embodiments, the AAV serotype is AAV8. In someembodiments, the AAV serotype is AAV9. In some embodiments, the AAVserotype is AAV12. In some embodiments, the AAV serotype is AAVrh.74.AAVs can also be self-complementary such that they do not requiresecond-strand DNA synthesis in the host cell (McCarty et al. (2001) GeneTher. 8:1248-54). Polynucleotides delivered by recombinant AAVs caninclude left (5′) and right (3′) inverted terminal repeats as part ofthe viral genome. In some embodiments, the recombinant viruses areinjected directly into target tissues. In alternative embodiments, therecombinant viruses are delivered systemically via the circulatorysystem.

In one embodiment, a recombinant virus used for meganuclease genedelivery is a self-limiting recombinant virus. A self-limiting virus canhave limited persistence time in a cell or organism due to the presenceof a recognition sequence for an engineered meganuclease within theviral genome. Thus, a self-limiting recombinant virus can be engineeredto provide a coding sequence for a promoter, an engineered meganucleasedescribed herein, and a meganuclease recognition site within the ITRs.The self-limiting recombinant virus delivers the meganuclease gene to acell, tissue, or organism, such that the meganuclease is expressed andable to cut the genome of the cell at an endogenous recognition sequencewithin the genome. The delivered meganuclease will also find its targetsite within the self-limiting recombinant viral genome, and cut therecombinant viral genome at this target site. Once cut, the 5′ and 3′ends of the viral genome will be exposed and degraded by exonucleases,thus killing the virus and ceasing production of the meganuclease.

If a polynucleotide comprising a nucleic acid sequence encoding anengineered meganuclease described herein is delivered to a cell by arecombinant virus (e.g. an AAV), the nucleic acid sequence encoding theengineered meganuclease can be operably linked to a promoter. In someembodiments, this can be a viral promoter such as endogenous promotersfrom the recombinant virus (e.g. the LTR of a lentivirus) or thewell-known cytomegalovirus- or SV40 virus-early promoters. In particularembodiments, nucleic acid sequences encoding the engineeredmeganucleases are operably linked to a promoter that drives geneexpression preferentially in the target cells (e.g., muscle cells ormuscle precursor cells). Examples of muscle-specific tissue promotersinclude but are not limited to those muscle-specific promoterspreviously described, including C5-12, the muscle-specific creatinekinase (MCK) promoter, or the smooth muscle 22 (SM22) promoter. In someembodiments, the muscle-specific promoter comprises the sequenceaccording to any one of SEQ ID NOs: 169-181. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a C5-12 promoter comprisingSEQ ID NO: 169. In some embodiments, the muscle-specific promotercomprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a murine MCK promoter comprising SEQ ID NO: 170. Insome embodiments, the muscle-specific promoter comprises a sequence 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a humanMCK promoter comprising SEQ ID NO: 171. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a MCK enhancer comprisingSEQ ID NO: 172. In some embodiments, the muscle-specific promotercomprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a modified MCK enhancer comprising SEQ ID NO: 173. Insome embodiments, the muscle-specific promoter comprises a sequence 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a spc5-12 promoter comprising SEQ ID NO: 174. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a MHCK7 promoter comprisingSEQ ID NO: 175. In some embodiments, the muscle-specific promotercomprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a CK8 promoter comprising SEQ ID NO: 176. In someembodiments, the muscle-specific promoter comprises a sequence 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a SK-CRM4promoter comprising SEQ ID NO: 177. In some embodiments, themuscle-specific promoter comprises a sequence 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a SP-301 promotercomprising SEQ ID NO: 178. In some embodiments, the muscle-specificpromoter comprises a sequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to a SP-817 promoter comprising SEQ ID NO:179. In some embodiments, the muscle-specific promoter comprises asequence 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to a SP-905 promoter comprising SEQ ID NO: 180. In someembodiments, the muscle-specific promoter comprises a sequence 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a musclehybrid promoter comprising SEQ ID NO: 181. In some embodiments, whereina single polynucleotide comprises two separate nucleic acid sequenceseach encoding an engineered meganuclease described herein, themeganuclease genes are operably linked to two separate promoters. Inalternative embodiments, the two meganuclease genes are operably linkedto a single promoter, and in some examples can be separated by aninternal-ribosome entry site (IRES) or a 2A peptide sequence (Szymezak &Vignali (2005) Expert Opin Biol Ther. 5:627-38). Such 2A peptidesequences can include, for example, a T2A, P2A, E2A, or F2A sequence.

In some embodiments, the methods include delivering an engineeredmeganuclease described herein, or a polynucleotide encoding the same, toa cell in combination with a second polynucleotide comprising anexogenous nucleic acid sequence encoding a sequence of interest, whereinthe engineered meganuclease is expressed in the cells, recognizes andcleaves a recognition sequence described herein (e.g., SEQ ID NO: 6, SEQID NO: 10, or SEQ ID NO: 12) within a dystrophin gene of the cell, andgenerates a cleavage site, wherein the exogenous nucleic acid andsequence of interest are inserted into the genome at the cleavage site(e.g., by homologous recombination). In some such examples, thepolynucleotide can comprise sequences homologous to nucleic acidsequences flanking the meganuclease cleavage site in order to promotehomologous recombination of the exogenous nucleic acid and sequence ofinterest into the genome.

Such polynucleotides comprising exogenous nucleic acids can beintroduced into a cell and/or delivered to a target cell in a subject byany of the means previously discussed. In particular embodiments, suchpolynucleotides comprising exogenous nucleic acid molecules areintroduced by way of a recombinant virus (i.e., a viral vector), such asa recombinant lentivirus, recombinant retrovirus, recombinantadenovirus, or a recombinant AAV. Recombinant AAVs useful forintroducing a polynucleotide comprising an exogenous nucleic acidmolecule can have any serotype (i.e., capsid) that allows fortransduction of the virus into the cell and insertion of the exogenousnucleic acid molecule sequence into the cell genome. In someembodiments, recombinant AAVs have a serotype of AAV1, AAV2, AAV5 AAV6,AAV7, AAV8, AAV9, AAV12, or AAVrh.74. In some embodiments, the AAVserotype is AAV1. In some embodiments, the AAV serotype is AAV2. In someembodiments, the AAV serotype is AAV5. In some embodiments, the AAVserotype is AAV6. In some embodiments, the AAV serotype is AAV7. In someembodiments, the AAV serotype is AAV8. In some embodiments, the AAVserotype is AAV9. In some embodiments, the AAV serotype is AAV12. Insome embodiments, the AAV serotype is AAVrh.74. The recombinant AAV canalso be self-complementary such that it does not require second-strandDNA synthesis in the host cell. Exogenous nucleic acid moleculesintroduced using a recombinant AAV can be flanked by a 5′ (left) and 3′(right) inverted terminal repeat in the viral genome.

In another particular embodiment, an exogenous nucleic acid molecule canbe introduced into a cell using a single-stranded DNA template. Thesingle-stranded DNA can comprise the exogenous nucleic acid moleculeand, in particular embodiments, can comprise 5′ and 3′ homology arms topromote insertion of the nucleic acid sequence into the nucleasecleavage site by homologous recombination. The single-stranded DNA canfurther comprise a 5′ AAV ITR sequence 5′ upstream of the 5′ homologyarm, and a 3′ AAV ITR sequence 3′ downstream of the 3′ homology arm.

In another particular embodiment, genes encoding a nuclease of theinvention and/or an exogenous nucleic acid molecule of the invention canbe introduced into a cell by transfection with a linearized DNAtemplate. A plasmid DNA encoding an engineered nuclease and/or anexogenous nucleic acid molecule can, for example, be digested by one ormore restriction enzymes such that the circular plasmid DNA islinearized prior to transfection into the cell.

When delivered to a cell, an exogenous nucleic acid of the invention canbe operably linked to any promoter suitable for expression of theencoded polypeptide in the cell, including those mammalian promoters andinducible promoters previously discussed. An exogenous nucleic acid ofthe invention can also be operably linked to a synthetic promoter.Synthetic promoters can include, without limitation, the JeT promoter(WO 2002/012514). In specific embodiments, a nucleic acid sequenceencoding an engineered meganuclease as disclosed herein can be operablylinked to a muscle-specific promoter discussed herein.

Administration

The target tissue(s) or target cell(s) include, without limitation,muscle cells, such as skeletal muscle cells, cardiac muscle cells, ormuscle cells of the diaphragm. In some embodiments, the target cell is amuscle progenitor cell such as a skeletal muscle progenitor cell or acardiac muscle progenitor cell. Such muscle progenitor cells have beendescribed in the art and can either be present in a subject or derivedfrom another stem cell population such as an induced pluripotent stemcell or an embryonic stem cell (Tey et al. (2019) Front. Cell Dev. Biol.7:284 and Amini et al. (2017) J. Cardiovasc. Thorac. Res. 9:127-32).

In some embodiments, engineered meganucleases described herein, orpolynucleotides encoding the same, are delivered to a cell in vitro. Insome embodiments, engineered meganucleases described herein, orpolynucleotides encoding the same, are delivered to a cell in a subjectin vivo. As discussed herein, meganucleases of the invention can bedelivered as purified protein or as a polynucleotide (e.g., RNA or DNA)comprising a nucleic acid sequence encoding the meganuclease. In someembodiments, meganuclease proteins, or polynucleotides encodingmeganucleases, are supplied to target cells (e.g., a muscle cell ormuscle progenitor cell) via injection directly to the target tissue.Alternatively, meganuclease proteins, or polynucleotides encodingmeganucleases, can be delivered systemically via the circulatory system.

In various embodiments of the methods, compositions described herein,such as the engineered meganucleases described herein, polynucleotidesencoding the same, recombinant viruses comprising such polynucleotides,or lipid nanoparticles comprising such polynucleotides, can beadministered via any suitable route of administration known in the art.Such routes of administration can include, for example, intravenous,intramuscular, intraperitoneal, subcutaneous, intrahepatic,transmucosal, transdermal, intraarterial, and sublingual. In someembodiments, the engineered meganuclease proteins, polynucleotidesencoding the same, recombinant viruses comprising such polynucleotides,or lipid nanoparticles comprising such polynucleotides, are supplied totarget cells (e.g., muscle cells or muscle precursor cells) viainjection directly to the target tissue (e.g., muscle tissue). Othersuitable routes of administration can be readily determined by thetreating physician as necessary.

In some embodiments, a therapeutically effective amount of an engineerednuclease described herein, or a polynucleotide encoding the same, isadministered to a subject in need thereof for the treatment of adisease. As appropriate, the dosage or dosing frequency of theengineered meganuclease, or the polynucleotide encoding the same, may beadjusted over the course of the treatment, based on the judgment of theadministering physician. Appropriate doses will depend, among otherfactors, on the specifics of any AAV chosen (e.g., serotype, etc.), anylipid nanoparticle chosen, on the route of administration, on thesubject being treated (i.e., age, weight, sex, and general condition ofthe subject), and the mode of administration. Thus, the appropriatedosage may vary from patient to patient. An appropriate effective amountcan be readily determined by one of skill in the art or treatingphysician. Dosage treatment may be a single dose schedule or, ifmultiple doses are required, a multiple dose schedule. Moreover, thesubject may be administered as many doses as appropriate. One of skillin the art can readily determine an appropriate number of doses. Thedosage may need to be adjusted to take into consideration an alternativeroute of administration or balance the therapeutic benefit against anyside effects.

In some embodiments, the methods further include administration of apolynucleotide comprising a nucleic acid sequence encoding asecretion-impaired hepatotoxin, or encoding tPA, which stimulateshepatocyte regeneration without acting as a hepatotoxin.

In some embodiments, a subject is administered a pharmaceuticalcomposition comprising a polynucleotide comprising a nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe encoding nucleic acid sequence is administered at a dose of about1×10¹⁰ gc/kg to about 1×10¹⁴ gc/kg (e.g., about 1×10¹⁰ gc/kg, about1×10¹¹ gc/kg, about 1×10¹² gc/kg, about 1×10¹³ gc/kg, or about 1×10¹⁴gc/kg). In some embodiments, a subject is administered a pharmaceuticalcomposition comprising a polynucleotide comprising a nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe encoding nucleic acid sequence is administered at a dose of about1×10¹⁰ gc/kg, about 1×10¹¹ gc/kg, about 1×10¹² gc/kg, about 1×10¹³gc/kg, or about 1×10¹⁴ gc/kg. In some embodiments, a subject isadministered a pharmaceutical composition comprising a polynucleotidecomprising a nucleic acid sequence encoding an engineered meganucleasedescribed herein, wherein the encoding nucleic acid sequence isadministered at a dose of about 1×10¹⁰ gc/kg to about 1×10¹¹ gc/kg,about 1×10¹¹ gc/kg to about 1×10¹² gc/kg, about 1×10¹² gc/kg to about1×10¹³ gc/kg, or about 1×10¹³ gc/kg to about 1×10¹⁴ gc/kg. It should beunderstood that these doses can relate to the administration of a singlepolynucleotide comprising a single nucleic acid sequence encoding asingle engineered meganuclease described herein or, alternatively, canrelate to a single polynucleotide comprising a first nucleic acidsequence encoding a first engineered meganuclease described herein and asecond nucleic acid sequence encoding a second engineered meganucleasedescribed herein, wherein each of the two encoding nucleic acidsequences is administered at the indicated dose.

In some embodiments, a subject is administered a lipid nanoparticleformulation comprising an mRNA comprising a nucleic acid sequenceencoding an engineered meganuclease described herein, wherein the doseof the mRNA is about 0.1 mg/kg to about 3 mg/kg. In some embodiments, asubject is administered a lipid nanoparticle formulation comprising anmRNA comprising a nucleic acid sequence encoding an engineeredmeganuclease described herein, wherein the dose of the mRNA is about 0.1mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, or about 3.0mg/kg. In some embodiments, a subject is administered a lipidnanoparticle formulation comprising an mRNA comprising a nucleic acidsequence encoding an engineered meganuclease described herein, whereinthe dose of the mRNA is about 0.1 mg/kg to about 0.25 mg/kg, about 0.25mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about0.75 mg/kg to about 1.0 mg/kg, about 1.0 mg/kg to about 1.5 mg/kg, about1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, orabout 2.5 mg/kg to about 3.0 mg/kg.

2.4 Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an engineeredmeganuclease described herein, or a pharmaceutically acceptable carrierand a polynucleotide described herein that comprises a nucleic acidsequence encoding an engineered meganuclease described herein. Suchpolynucleotides can be, for example, mRNA or DNA as described herein. Insome such examples, the polynucleotide in the pharmaceutical compositioncan be comprised by a lipid nanoparticle or can be comprised by arecombinant virus (e.g., a recombinant AAV). In other embodiments, thedisclosure provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and a genetically modified cell ofthe invention, which can be delivered to a target tissue where the cellexpresses the engineered meganuclease as disclosed herein. Suchpharmaceutical compositions are formulated, for example, for systemicadministration, or administration to target tissues.

In various embodiments of the invention, the pharmaceutical compositionscan be useful for treating DMD, converting a DMD disease phenotype to aBecker Muscular Dystrophy phenotype, and/or reducing the symptomsassociated with DMD in a subject.

Such pharmaceutical compositions can be prepared in accordance withknown techniques. See, e.g., Remington, The Science And Practice ofPharmacy (21st ed., Philadelphia, Lippincott, Williams & Wilkins, 2005).In the manufacture of a pharmaceutical formulation according to theinvention, engineered meganucleases described herein, polynucleotidesencoding the same, or cells expressing the same, are typically admixedwith a pharmaceutically acceptable carrier and the resulting compositionis administered to a subject. The carrier must be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the subject. The carrier can be a solidor a liquid, or both, and can be formulated with the compound as aunit-dose formulation.

In some embodiments, pharmaceutical compositions of the invention canfurther comprise one or more additional agents or biological moleculesuseful in the treatment of a disease in the subject. Likewise, theadditional agent(s) and/or biological molecule(s) can be co-administeredas a separate composition.

The pharmaceutical compositions described herein can include atherapeutically effective amount of any engineered meganucleasedisclosed herein, or any polynucleotide described herein encoding anyengineered meganuclease described herein. For example, in someembodiments, the pharmaceutical composition can include polynucleotidesdescribed herein at any of the doses (e.g., gc/kg of an encoding nucleicacid sequence or mg/kg of mRNA) described herein.

In particular embodiments of the invention, the pharmaceuticalcomposition can comprise one or more recombinant viruses (e.g.,recombinant AAVs) described herein that comprise one or morepolynucleotides described herein (i.e., packaged within the viralgenome). In particular embodiments, the pharmaceutical compositioncomprises two or more recombinant viruses (e.g., recombinant AAVs)described herein, each comprising a polynucleotide comprising a nucleicacid sequence encoding a different engineered meganuclease describedherein. For example, a first recombinant virus (e.g., recombinant AAV)may comprise a first polynucleotide comprising a first nucleic acidsequence encoding a first engineered meganuclease described hereinhaving specificity for the DMD 19-20 recognition sequence, and a secondrecombinant virus (e.g., recombinant AAV) comprising a secondpolynucleotide comprising a second nucleic acid sequence encoding asecond engineered meganuclease described herein having specificity forthe DMD 35-36 recognition sequence or the DMD 37-38 recognitionsequence. The expression of such a pair of engineered meganucleases inthe same cell (e.g., a muscle cell) would allow for the excision ofexons 45-55 from the dystrophin gene according to the invention.

In other particular embodiments, the pharmaceutical composition cancomprise a recombinant virus (e.g., recombinant AAV) described hereinthat comprises a polynucleotide (i.e., packaged within the viral genome)that comprises two nucleic acid sequences encoding two separateengineered meganucleases described herein. For example, the recombinantvirus (e.g., recombinant AAV) can comprise a polynucleotide comprising afirst nucleic acid sequence encoding a first engineered meganucleasedescribed herein having specificity for the DMD 19-20 recognitionsequence, and a second nucleic acid sequence encoding a secondengineered meganuclease described herein having specificity for the DMD35-36 recognition sequence or the DMD 37-38 recognition sequence. Theexpression of such a pair of engineered meganucleases would allow forthe excision of exons 45-55 from the dystrophin gene according to theinvention.

In particular embodiments of the invention, the pharmaceuticalcomposition can comprise one or more polynucleotides (e.g., mRNAs)described herein encapsulated within lipid nanoparticles. In particularembodiments, lipid nanoparticles can comprise two or morepolynucleotides (e.g., mRNAs) described herein, each comprising anucleic acid sequence encoding a different engineered meganucleasedescribed herein. For example, a first polynucleotide (e.g., mRNA) inthe lipid nanoparticle may encode a first engineered meganucleasedescribed herein having specificity for the DMD 19-20 recognitionsequence, and a second polynucleotide (e.g., mRNA) in the lipidnanoparticle may encode a second engineered meganuclease describedherein having specificity for the DMD 35-36 recognition sequence or theDMD 37-38 recognition sequence. The expression of such a pair ofengineered meganucleases in the same cell (e.g., a muscle cell) wouldallow for the excision of exons 45-55 from the dystrophin gene accordingto the invention. Alternatively, the pharmaceutical composition cancomprise two separate populations of lipid nanoparticles, eachcomprising a different polynucleotide (e.g., mRNA) described herein,wherein a first population of lipid nanoparticles comprise a firstpolynucleotide (e.g., mRNA) described herein encoding a first engineeredmeganuclease having specificity for the DMD 19-20 recognition sequence,and the second population of lipid nanoparticles comprise a secondpolynucleotide (e.g., mRNA) described herein encoding a secondengineered meganuclease having specificity for the DMD 35-36 recognitionsequence or the DMD 37-38 recognition sequence.

In other particular embodiments, lipid nanoparticles can comprise onepolynucleotide (e.g., mRNA) described herein that comprises two nucleicacid sequences encoding two separate engineered meganucleases describedherein. For example, the lipid nanoparticle can comprise apolynucleotide (e.g., mRNA) comprising a first nucleic acid sequenceencoding a first engineered meganuclease described herein havingspecificity for the DMD 19-20 recognition sequence, and a second nucleicacid sequence encoding a second engineered meganuclease described hereinhaving specificity for the DMD 35-36 recognition sequence or the DMD37-38 recognition sequence. The expression of such a pair of engineeredmeganucleases in the same cell (e.g., a muscle cell) would allow for theexcision of exons 45-55 from the dystrophin gene according to theinvention.

Some lipid nanoparticles contemplated for use in the invention compriseat least one cationic lipid, at least one non-cationic lipid, and atleast one conjugated lipid. In more particular examples, lipidnanoparticles can comprise from about 50 mol % to about 85 mol % of acationic lipid, from about 13 mol % to about 49.5 mol % of anon-cationic lipid, and from about 0.5 mol % to about 10 mol % of alipid conjugate, and are produced in such a manner as to have anon-lamellar (i.e., non-bilayer) morphology. In other particularexamples, lipid nanoparticles can comprise from about 40 mol % to about85 mol % of a cationic lipid, from about 13 mol % to about 49.5 mol % ofa non-cationic lipid, and from about 0.5 mol % to about 10 mol % of alipid conjugate and are produced in such a manner as to have anon-lamellar (i.e., non-bilayer) morphology.

Cationic lipids can include, for example, one or more of the following:palmitoyi-oleoyl-nor-arginine (PONA), MPDACA, GUADACA,((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) (MC3), LenMC3, CP-LenMC3, γ-LenMC3,CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3,Pan-MC4 and Pan MC5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane(DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), ormixtures thereof. The cationic lipid can also be DLinDMA, DLin-K-C2-DMA(“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC,MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3, Pan-MC4, Pan MC5, or mixturesthereof.

In various embodiments, the cationic lipid may comprise from about 50mol % to about 90 mol %, from about 50 mol % to about 85 mol %, fromabout 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %,from about 50 mol % to about 70 mol %, from about 50 mol % to about 65mol %, or from about 50 mol % to about 60 mol % of the total lipidpresent in the particle.

In other embodiments, the cationic lipid may comprise from about 40 mol% to about 90 mol %, from about 40 mol % to about 85 mol %, from about40 mol % to about 80 mol %, from about 40 mol % to about 75 mol %, fromabout 40 mol % to about 70 mol %, from about 40 mol % to about 65 mol %,or from about 40 mol % to about 60 mol % of the total lipid present inthe particle.

The non-cationic lipid may comprise, e.g., one or more anionic lipidsand/or neutral lipids. In particular embodiments, the non-cationic lipidcomprises one of the following neutral lipid components: (1) cholesterolor a derivative thereof; (2) a phospholipid; or (3) a mixture of aphospholipid and cholesterol or a derivative thereof. Examples ofcholesterol derivatives include, but are not limited to, cholestanol,cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethylether, cholesteryl-4′-hydroxybutyl ether, and mixtures thereof. Thephospholipid may be a neutral lipid including, but not limited to,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine(EPC), and mixtures thereof. In certain embodiments, the phospholipid isDPPC, DSPC, or mixtures thereof.

In some embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol% to about 60 mol %, from about 25 mol % to about 60 mol %, from about30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, fromabout 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %,from about 25 mol % to about 55 mol %, from about 30 mol % to about 55mol %, from about 13 mol % to about 50 mol %, from about 15 mol % toabout 50 mol % or from about 20 mol % to about 50 mol % of the totallipid present in the particle. When the non-cationic lipid is a mixtureof a phospholipid and cholesterol or a cholesterol derivative, themixture may comprise up to about 40, 50, or 60 mol % of the total lipidpresent in the particle.

The conjugated lipid that inhibits aggregation of particles maycomprise, e.g., one or more of the following: a polyethyleneglycol(PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, acationic-polymer-lipid conjugates (CPLs), or mixtures thereof. In oneparticular embodiment, the nucleic acid-lipid particles comprise eithera PEG-lipid conjugate or an ATTA-lipid conjugate. In certainembodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is usedtogether with a CPL. The conjugated lipid that inhibits aggregation ofparticles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol(DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide(Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), aPEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), ormixtures thereof.

Additional PEG-lipid conjugates suitable for use in the inventioninclude, but are not limited to,mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). Thesynthesis of PEG-C-DOMG is described in WO 2009/086558. Yet additionalPEG-lipid conjugates suitable for use in the invention include, withoutlimitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969.

In some cases, the conjugated lipid that inhibits aggregation ofparticles (e.g., PEG-lipid conjugate) may comprise from about 0.1 mol %to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, fromabout 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % toabout 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, orabout 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. Typically, in such instances, the PEG moiety has an averagemolecular weight of about 2,000 Daltons. In other cases, the conjugatedlipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate)may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol %to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol% to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol%, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.Typically, in such instances, the PEG moiety has an average molecularweight of about 750 Daltons.

In other embodiments, the composition may comprise amphoteric liposomes,which contain at least one positive and at least one negative chargecarrier, which differs from the positive one, the isoelectric point ofthe liposomes being between 4 and 8. This objective is accomplishedowing to the fact that liposomes are prepared with a pH-dependent,changing charge.

Liposomal structures with the desired properties are formed, forexample, when the amount of membrane-forming or membrane-based cationiccharge carriers exceeds that of the anionic charge carriers at a low pHand the ratio is reversed at a higher pH. This is always the case whenthe ionizable components have a pKa value between 4 and 9. As the pH ofthe medium drops, all cationic charge carriers are charged more and allanionic charge carriers lose their charge.

Cationic compounds useful for amphoteric liposomes include thosecationic compounds previously described herein above. Withoutlimitation, strongly cationic compounds can include, for example:DC-Chol 3-β-[N—(N′,N′-dimethylmethane) carbamoyl] cholesterol, TC-Chol3-β-[N—(N′, N′, N′-trimethylaminoethane) carbamoyl cholesterol, BGSCbisguanidinium-spermidine-cholesterol, BGTCbis-guadinium-tren-cholesterol, DOTAP(1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride, DOSPER(1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylarnide, DOTMA(1,2-dioleoyloxypropyl)-N,N,N-trimethylamronium chloride) (Lipofectin®),DORIE 1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide,DOSC (1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester), DOGSDSO(1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide omithine),DDAB dimethyldioctadecylammonium bromide, DOGS ((C18)2GlySper3+)N,N-dioctadecylamido-glycol-spermin (Transfectam®)(C18)2Gly+N,N-dioctadecylamido-glycine, CTAB cetyltrimethylammoniumbromide, CpyC cetylpyridinium chloride, DOEPC1,2-dioleoly-sn-glycero-3-ethylphosphocholine or otherO-alkyl-phosphatidylcholine or ethanolamines, amides from lysine,arginine or ornithine and phosphatidyl ethanolamine.

Examples of weakly cationic compounds include, without limitation:His-Chol (histaminyl-cholesterol hemisuccinate), Mo-Chol(morpholine-N-ethylamino-cholesterol hemisuccinate), or histidinyl-PE.

Examples of neutral compounds include, without limitation: cholesterol,ceramides, phosphatidyl cholines, phosphatidyl ethanolamines, tetraetherlipids, or diacyl glycerols.

Anionic compounds useful for amphoteric liposomes include thosenon-cationic compounds previously described herein. Without limitation,examples of weakly anionic compounds can include: CCHEMS (cholesterolhemisuccinate), alkyl carboxylic acids with 8 to 25 carbon atoms, ordiacyl glycerol hemisuccinate. Additional weakly anionic compounds caninclude the amides of aspartic acid, or glutamic acid and PE as well asPS and its amides with glycine, alanine, glutamine, asparagine, serine,cysteine, threonine, tyrosine, glutamic acid, aspartic acid or otheramino acids or aminodicarboxylic acids. According to the same principle,the esters of hydroxycarboxylic acids or hydroxydicarboxylic acids andPS are also weakly anionic compounds.

In some embodiments, amphoteric liposomes may contain a conjugatedlipid, such as those described herein above. Particular examples ofuseful conjugated lipids include, without limitation, PEG-modifiedphosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates(e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines andPEG-modified 1,2-diacyloxypropan-3-amines. Particular examples arePEG-modified diacylglycerols and dialkylglycerols.

In some embodiments, the neutral lipids may comprise from about 10 mol %to about 60 mol %, from about 15 mol % to about 60 mol %, from about 20mol % to about 60 mol %, from about 25 mol % to about 60 mol %, fromabout 30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %,from about 15 mol % to about 55 mol %, from about 20 mol % to about 55mol %, from about 25 mol % to about 55 mol %, from about 30 mol % toabout 55 mol %, from about 13 mol % to about 50 mol %, from about 15 mol% to about 50 mol % or from about 20 mol % to about 50 mol % of thetotal lipid present in the particle.

In some cases, the conjugated lipid that inhibits aggregation ofparticles (e.g., PEG-lipid conjugate) may comprise from about 0.1 mol %to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, fromabout 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % toabout 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, orabout 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. Typically, in such instances, the PEG moiety has an averagemolecular weight of about 2,000 Daltons. In other cases, the conjugatedlipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate)may comprise from about 5.0 mol % to about 10 mol %, from about 5 mol %to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol% to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol%, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.Typically, in such instances, the PEG moiety has an average molecularweight of about 750 Daltons.

Considering the total amount of neutral and conjugated lipids, theremaining balance of the amphoteric liposome can comprise a mixture ofcationic compounds and anionic compounds formulated at various ratios.The ratio of cationic to anionic lipid may selected in order to achievethe desired properties of nucleic acid encapsulation, zeta potential,pKa, or other physicochemical property that is at least in partdependent on the presence of charged lipid components.

In some embodiments, the lipid nanoparticles have a composition, whichspecifically enhances delivery and uptake in the liver, and specificallywithin hepatocytes.

In some embodiments, pharmaceutical compositions of the invention canfurther comprise one or more additional agents useful in the treatmentof DMD in the subject.

The present disclosure also provides engineered meganucleases describedherein, or polynucleotides described herein encoding the same, or cellsdescribed herein expressing engineered meganucleases described hereinfor use as a medicament. The present disclosure further provides the useof engineered meganucleases described herein, or polynucleotidesdisclosed herein encoding the same, or cells described herein expressingengineered meganucleases described herein in the manufacture of amedicament for treating DMD, for increasing levels of a modifieddystrophin protein (i.e., lacking the amino acids encoded by exons 45-55of the dystrophin gene), or reducing the symptoms associated with DMD.

2.5 Methods for Producing Recombinant Viruses

In some embodiments, the invention provides recombinant viruses, such asrecombinant AAVs, for use in the methods of the invention. RecombinantAAVs are typically produced in mammalian cell lines such as HEK-293.Because the viral cap and rep genes are removed from the recombinantvirus to prevent its self-replication to make room for the therapeuticgene(s) to be delivered (e.g., the meganuclease gene), it is necessaryto provide these in trans in the packaging cell line. In addition, it isnecessary to provide the “helper” (e.g., adenoviral) componentsnecessary to support replication (Cots et al. (2013) Curr. Gene Ther.13:370-81). Frequently, recombinant AAVs are produced using atriple-transfection in which a cell line is transfected with a firstplasmid encoding the “helper” components, a second plasmid comprisingthe cap and rep genes, and a third plasmid comprising the viral ITRscontaining the intervening DNA sequence to be packaged into the virus.Viral particles comprising a genome (ITRs and intervening gene(s) ofinterest) encased in a capsid are then isolated from cells byfreeze-thaw cycles, sonication, detergent, or other means known in theart. Particles are then purified using cesium-chloride density gradientcentrifugation or affinity chromatography and subsequently delivered tothe gene(s) of interest to cells, tissues, or an organism such as ahuman patient.

Because recombinant AAV particles are typically produced (manufactured)in cells, precautions must be taken in practicing the current inventionto ensure that the engineered meganuclease is not expressed in thepackaging cells. Because the recombinant viral genomes of the inventionmay comprise a recognition sequence for the meganuclease, anymeganuclease expressed in the packaging cell line may be capable ofcleaving the viral genome before it can be packaged into viralparticles. This will result in reduced packaging efficiency and/or thepackaging of fragmented genomes. Several approaches can be used toprevent meganuclease expression in the packaging cells.

The nuclease can be placed under the control of a tissue-specificpromoter that is not active in the packaging cells. Any tissue specificpromoter described herein for expression of the engineered meganucleaseor for a nucleic acid sequence of interest can be used. For example, ifa recombinant virus is developed for delivery of genes encoding anengineered meganuclease to muscle tissue, a muscle-specific promoter canbe used. Examples of muscle-specific promoters include, withoutlimitation, those muscle-specific promoters described elsewhere herein.

Alternatively, the recombinant virus can be packaged in cells from adifferent species in which the meganuclease is not likely to beexpressed. For example, viral particles can be produced in microbial,insect, or plant cells using mammalian promoters, such as the well-knowncytomegalovirus- or SV40 virus-early promoters, which are not active inthe non-mammalian packaging cells. In a particular embodiment, viralparticles are produced in insect cells using the baculovirus system asdescribed by Gao et al. (2007) J. Biotechnol. 131:138-43. A meganucleaseunder the control of a mammalian promoter is unlikely to be expressed inthese cells (Airenne et al. (2013) Mol. Ther. 21:739-49). Moreover,insect cells utilize different mRNA splicing motifs than mammaliancells. Thus, it is possible to incorporate a mammalian intron, such asthe human growth hormone (HGH) intron or the SV40 large T antigenintron, into the coding sequence of a meganuclease. Because theseintrons are not spliced efficiently from pre-mRNA transcripts in insectcells, insect cells will not express a functional meganuclease and willpackage the full-length genome. In contrast, mammalian cells to whichthe resulting recombinant AAV particles are delivered will properlysplice the pre-mRNA and will express functional meganuclease protein.Chen has reported using HGH and SV40 large T antigen introns toattenuate expression of the toxic proteins barnase and diphtheria toxinfragment A in insect packaging cells, enabling the production ofrecombinant AAV vectors carrying these toxin genes (Chen (2012) Mol.Ther. Nucleic Acids. 1: e57).

The engineered meganuclease gene can be operably linked to an induciblepromoter such that a small-molecule inducer is required for meganucleaseexpression. Examples of inducible promoters include the Tet-On system(Clontech; Chen et al. (2015) BMC Biotechnol. 15:4) and the RheoSwitchsystem (Intrexon; Sowa i (2011) Spine 36:E623-8). Both systems, as wellas similar systems known in the art, rely on ligand-inducibletranscription factors (variants of the Tet Repressor and Ecdysonereceptor, respectively) that activate transcription in response to asmall-molecule activator (Doxycycline or Ecdysone, respectively).Practicing the current invention using such ligand-inducibletranscription activators includes: 1) placing the engineeredmeganuclease gene under the control of a promoter that responds to thecorresponding transcription factor, the meganuclease gene having (a)binding site(s) for the transcription factor; and 2) including the geneencoding the transcription factor in the packaged viral genome. Thelatter step is necessary because the engineered meganuclease will not beexpressed in the target cells or tissues following recombinant AAVdelivery if the transcription activator is not also provided to the samecells. The transcription activator then induces meganuclease geneexpression only in cells or tissues that are treated with the cognatesmall-molecule activator. This approach is advantageous because itenables meganuclease gene expression to be regulated in aspatio-temporal manner by selecting when and to which tissues thesmall-molecule inducer is delivered. However, the requirement to includethe inducer in the viral genome, which has significantly limitedcarrying capacity, creates a drawback to this approach.

In another particular embodiment, recombinant AAV particles are producedin a mammalian cell line that expresses a transcription repressor thatprevents expression of the meganuclease. Transcription repressors areknown in the art and include the Tet-Repressor, the Lac-Repressor, theCro repressor, and the Lambda-repressor. Many nuclear hormone receptorssuch as the ecdysone receptor also act as transcription repressors inthe absence of their cognate hormone ligand. To practice the currentinvention, packaging cells are transfected/transduced with a vectorencoding a transcription repressor and the meganuclease gene in theviral genome (packaging vector) is operably linked to a promoter that ismodified to comprise binding sites for the repressor such that therepressor silences the promoter. The gene encoding the transcriptionrepressor can be placed in a variety of positions. It can be encoded ona separate vector; it can be incorporated into the packaging vectoroutside of the ITR sequences; it can be incorporated into the cap/repvector or the adenoviral helper vector, or it can be stably integratedinto the genome of the packaging cell such that it is expressedconstitutively. Methods to modify common mammalian promoters toincorporate transcription repressor sites are known in the art. Forexample, Chang & Roninson modified the strong, constitutive CMV and RSVpromoters to comprise operators for the Lac repressor and showed thatgene expression from the modified promoters was greatly attenuated incells expressing the repressor (Chang & Roninson (1996) Gene183:137-42). The use of a non-human transcription repressor ensures thattranscription of the meganuclease gene will be repressed only in thepackaging cells expressing the repressor and not in target cells ortissues transduced with the resulting recombinant AAV.

2.6 Engineered Meganuclease Variants

Embodiments of the invention encompass the engineered meganucleasesdescribed herein, and variants thereof. Further embodiments of theinvention encompass polynucleotides comprising a nucleic acid sequenceencoding the engineered meganucleases described herein, and variants ofsuch polynucleotides.

As used herein, “variants” is intended to mean substantially similarsequences. A “variant” polypeptide is intended to mean a polypeptidederived from the “native” polypeptide by deletion or addition of one ormore amino acids at one or more internal sites in the native proteinand/or substitution of one or more amino acids at one or more sites inthe native polypeptide. As used herein, a “native” polynucleotide orpolypeptide comprises a parental sequence from which variants arederived. Variant polypeptides encompassed by the embodiments arebiologically active. That is, they continue to possess the desiredbiological activity of the native protein; i.e., the ability to bind andcleave a dystrophin gene recognition sequence described herein (e.g., aDMD 19-20, DMD 35-36, or DMD 37-38 recognition sequence). Such variantsmay result, for example, from human manipulation. Biologically activevariants of a native polypeptide of the embodiments (e.g., SEQ ID NOs:36-59), or biologically active variants of the recognition half-sitebinding subunits described herein, will have at least about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequenceidentity to the amino acid sequence of the native polypeptide, nativesubunit, native HVR1 region, and/or native HVR2 region, as determined bysequence alignment programs and parameters described elsewhere herein. Abiologically active variant of a polypeptide or subunit of theembodiments may differ from that polypeptide or subunit by as few asabout 1-40 amino acid residues, as few as about 1-20, as few as about1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, e.g., Kunkel (1985) Proc.Natl. Acad. Sci. USA 82:488-92; Kunkel et al. (1987) Methods Enzymol.154:367-82; U.S. Pat. No. 4,873,192; Walker & Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, New York)and the references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be optimal.

In some embodiments, engineered meganucleases of the invention cancomprise variants of the HVR1 and HVR2 regions disclosed herein.Parental HVR regions can comprise, for example, residues 24-79 orresidues 215-270 of the exemplified engineered meganucleases. Thus,variant HVRs can comprise an amino acid sequence having at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or more, sequence identity to an amino acid sequencecorresponding to residues 24-79 or residues 215-270 of the engineeredmeganucleases exemplified herein, such that the variant HVR regionsmaintain the biological activity of the engineered meganuclease (i.e.,binding to and cleaving the recognition sequence). Further, in someembodiments of the invention, a variant HVR1 region or variant HVR2region can comprise residues corresponding to the amino acid residuesfound at specific positions within the parental HVR. In this context,“corresponding to” means that an amino acid residue in the variant HVRis the same amino acid residue (i.e., a separate identical residue)present in the parental HVR sequence in the same relative position(i.e., in relation to the remaining amino acids in the parent sequence).By way of example, if a parental HVR sequence comprises a serine residueat position 26, a variant HVR that “comprises a residue correspondingto” residue 26 will also comprise a serine at a position that isrelative (i.e., corresponding) to parental position 26.

In particular embodiments, engineered meganucleases of the inventioncomprise an HVR1 that has at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more sequence identity to an amino acidsequence corresponding to residues 24-79 of any one of SEQ ID NOs:36-59.

In certain embodiments, engineered meganucleases of the inventioncomprise an HVR2 that has 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or more sequence identity to an amino acid sequencecorresponding to residues 215-270 of any one of SEQ ID NOs: 36-59.

A substantial number of amino acid modifications to the DNA recognitiondomain of the wild-type I-CreI meganuclease have previously beenidentified (e.g., U.S. Pat. No. 8,021,867), which singly or incombination, result in engineered meganucleases with specificitiesaltered at individual bases within the DNA recognition sequencehalf-site, such that the resulting rationally-designed meganucleaseshave half-site specificities different from the wild-type enzyme. Table9 provides potential substitutions that can be made in an engineeredmeganuclease monomer or subunit to enhance specificity based on the basepresent at each half-site position (−1 through −9) of a recognitionhalf-site.

TABLE 9 Favored Sense-Strand Base Posn. A C G T A/T A/C A/G C/T G/TA/G/T A/C/G/T -1 Y75 R70* K70 Q70* T46* G70 L75* H75* E70* C70 A70 C75*R75* E75* L70 S70 Y139* H46* E46* Y75* G46* C46* K46* D46* Q75* A46*R46* H75* H139 Q46* H46* -2 Q70 E70 H70 Q44* C44* T44* D70 D44* A44*K44* E44* V44* R44* I44* L44* N44* -3 Q68 E68 R68 M68 H68 Y68 K68 C24*F68 C68 I24* K24* L68 R24* F68 -4 A26* E77 R77 S77 S26* Q77 K26* E26*Q26* -5 E42 R42 K28* C28* M66 Q42 K66 -6 Q40 E40 R40 C40 A40 S40 C28*R28* I40 A79 S28* V40 A28* C79 H28* I79 V79 Q28* -7 N30* E38 K38 I38 C38H38 Q38 K30* R38 L38 N38 R30* E30* Q30* -8 F33 E33 F33 L33 R32* R33 Y33D33 H33 V33 I33 F33 C33 -9 E32 R32 L32 D32 S32 K32 V32 I32 N32 A32 H32C32 Q32 T32 Bold entries are wild-type contact residues and do notconstitute “modifications” as used herein. An asterisk indicates thatthe residue contacts the base on the antisense strand.

Certain modifications can be made in an engineered meganuclease monomeror subunit to modulate DNA-binding affinity and/or activity. Forexample, an engineered meganuclease monomer or subunit described hereincan comprise a G, S, or A at a residue corresponding to position 19 ofI-CreI or any one of SEQ ID NOs: 36-59 (WO 2009/001159), a Y, R, K, or Dat a residue corresponding to position 66 of I-CreI or any one of SEQ IDNOs: 36-59, and/or an E, Q, or K at a residue corresponding to position80 of I-CreI or any one of SEQ ID NOs: 36-59 (U.S. Pat. No. 8,021,867).

For polynucleotides, a “variant” comprises a deletion and/or addition ofone or more nucleotides at one or more sites within the nativepolynucleotide. One of skill in the art will recognize that variants ofthe nucleic acids of the embodiments will be constructed such that theopen reading frame is maintained. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the embodiments. Variant polynucleotides include syntheticallyderived polynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a recombinant nucleaseof the embodiments. Generally, variants of a particular polynucleotideof the embodiments will have at least about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99% or more sequence identity tothat particular polynucleotide as determined by sequence alignmentprograms and parameters described elsewhere herein. Variants of aparticular polynucleotide of the embodiments (i.e., the referencepolynucleotide) can also be evaluated by comparison of the percentsequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by screening the polypeptide its intendedactivity. For example, variants of an engineered meganuclease would bescreened for their ability to preferentially bind and cleave recognitionsequences found within a dystrophin gene.

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Characterization of Meganucleases that Bind and CleaveRecognition Sequences in the Dystrophin Gene 1. Meganucleases that Bindand Cleave the DMD 19-20, DMD 29-30, DMD 35-36, and DMD 37-38Recognition Sequences

These experiments were designed to utilize pairs of engineeredmeganucleases that allow for the removal of a segment of the dystrophingene. This strategy of employing two engineered meganucleases to edit attwo locations simultaneously allows for the excision of multiple exons,specifically exons 45 through 55. In some cases, two engineeredmeganucleases delivered on a single AAV vector can make double-strandedbreaks in the intron sequences upstream of exon 45 and downstream ofexon 55; i.e., the introns flanking exons 45-55 of the gene.

As described herein, the pairs of engineered meganucleases selected foruse are designed to cleave recognition sequences with identical 4 basepair center sequences. As a result, cleavage by the engineeredmeganucleases will leave complementary sticky ends (i.e., 4 base pair,3′ overhangs) so that the genomic sequence will be perfectly ligatedfollowing excision of the “hot spot” of exons 45-55, where over 50% ofDMD-causing mutations are found. Removal of these exons results in theexcision of over 500,000 base pairs of genomic sequence from the gene,returns the reading frame of the gene back to normal relative towild-type, and creates a shorter “Becker's” dystrophin that has beenshown to be therapeutically relevant in preclinical models. FIG. 1provides the approximate location of the recognition sequences and theresulting ligation of the intron between exon 44 and exon 56. Subsequentsplicing during transcription provides an mRNA transcript where exon 44is in frame with exon 56 resulting in the shortened but functionaldystrophin protein.

Hundreds of potential pairs of engineered meganucleases were evaluatedfor activity and efficacy. Of these, a select number were furthercharacterized for their ability to cleave within the dystrophin gene andgenerate a perfect ligation of the genetic material.

Engineered meganucleases designed to bind and cleave the DMD 19-20 (SEQID NO: 6), DMD 29-30 (SEQ ID NO: 8), DMD 35-36 (SEQ ID NO: 10), and DMD37-38 (SEQ ID NO: 12) recognition sequences within introns of thedystrophin gene are referred to herein as DMD 19-20 meganucleases, DMD29-30 meganucleases, DMD 35-36 meganucleases, and DMD 37-38meganucleases, respectively. The DMD 19-20 recognition sequence ispositioned in the intron 5′ upstream of exon 45. The DMD 29-30recognition sequence is positioned in the intron 5′ upstream of exon 46.Both the DMD 35-36 and DMD 37-38 recognition sequences are positioned inthe intron 3′ downstream of exon 55.

Each DMD engineered meganuclease comprises an N-terminal nuclearlocalization signal derived from SV40, a first meganuclease subunit, alinker sequence, and a second meganuclease subunit. A first subunit ineach DMD meganuclease binds to a first DMD recognition half-site, whilea second subunit binds to a second DMD recognition half-site. Forexample, a first subunit in the DMD 19-20 meganuclease binds to a firstDMD recognition half-site, while a second subunit binds to a second DMD19-20 recognition half-site (FIG. 1 ).

The DMD meganuclease subunits each comprise a 56 base pair hypervariableregion, referred to as HVR1 and HVR2, respectively. The HVR1 region ofeach DMD meganuclease binding subunit consists of residues 24-79 of SEQID NOs: 36-59. The HVR2 region of each DMD meganuclease consists ofresidues 215-270 of SEQ ID NOs: 36-59.

2. Cleavage of DMD 19-20, DMD 29-30, DMD 35-36, and DMD 37-38Recognition Sequences in a CHO Cell Reporter Assay

To determine whether the DMD 19-20, DMD 29-30, DMD 35-36, and DMD 37-38meganucleases could bind and cleave their respective human recognitionsequences, each engineered meganuclease was evaluated using the CHO cellreporter assay previously described (see, WO 2012/167192 and FIG. 5 ).To perform the assays, CHO cell reporter lines were produced, whichcarried a non-functional Green Fluorescent Protein (GFP) gene expressioncassette integrated into the genome of the cells. The GFP gene in eachcell line was interrupted by a pair of recognition sequences such thatintracellular cleavage of either recognition sequence by a meganucleasewould stimulate a homologous recombination event resulting in afunctional GFP gene.

In CHO reporter cell lines developed for this study, one recognitionsequence inserted into the GFP gene was the human DMD 19-20, DMD 29-30,DMD 35-36, and DMD 37-38 recognition sequences. The second recognitionsequence inserted into the GFP gene was a CHO-23/24 recognitionsequence, which is recognized and cleaved by a control meganucleasecalled “CHO-23/24.”

CHO reporter cells were transfected with mRNA encoding the DMD19-20x.13, DMD 19-20x.87, DMD 19-20L.249, DMD 19-20L.374, DMD19-20L.375, DMD 19-20L.431, DMD 19-20L.458, DMD 29-30x.18, DMD29-30x.40, DMD 35-36x.63, DMD 35-36x.81, DMD 35-36L.376, DMD 35-36L.457,DMD 35-36L.469, DMD 37-38x.15, DMD 37-38x.66, DMD 37-38x.79, DMD37-38L.166, DMD 37-38L.478, DMD 37-38L.512, and DMD 37-38L.528meganucleases. CHO reporter cells were also transfected with mRNAencoding the CHO-23/24 meganuclease. In each assay, 5e4 CHO reportercells were transfected with 90 ng of mRNA in a 96-well plate usingLipofectamine® MessengerMax (ThermoFisher) according to themanufacturer's instructions. The transfected CHO cells were evaluated byflow cytometry at 2 days, 5 days, and 7 days post transfection todetermine the percentage of GFP-positive cells compared to anuntransfected negative control. Data obtained at each time point wasnormalized to the % GFP positive cells observed using the CHO-23/24meganuclease to determine an “activity score,” and the normalized datafrom the earliest time point was subtracted from that of the latest timepoint to determine a “toxicity score.” The activity and toxicity scoreswere then added together to determine an “activity index,” which wasthen normalized to the activity index of the CHO-23/24 meganuclease tocompare data between cell lines (“normalized activity index”).

3. Results

As shown in FIG. 6A-6I, each of the respective meganucleases were ableto bind and cleave their respective recognition sequence. Eachmeganuclease provided a level of GFP expression that was as high orhigher than the positive control CHO 23-24 meganuclease.

4. Conclusions

These studies demonstrated that engineered meganucleases of theinvention could efficiently bind and cleave their respective humanrecognition sequences (e.g., recognition sequences of SEQ ID NOs: 6, 8,10, and 12) in cells.

Example 2 Characterization of Insertions and Deletions Induced by DMDMeganucleases 1. Methods

DMD 19-20, DMD 29-30, DMD 35-36, and DMD 37-38 meganucleases were eachevaluated for their ability to generate insertions or deletions(“indels”) at their respective recognition sequences in MRC5 cells.

In these experiments, 1e6 MRC5 cells were electroporated with 2 ng or100 ng mRNA encoding DMD meganucleases (DMD 19-20x.13, DMD 19-20x.87,DMD 29-30x.18, DMD 29-30x.40, DMD 35-36x.63, DMD 35-36x.81, DMD37-38x.15, DMD 37-38x.66, or DMD 37-38x.79), or GFP using the LonzaAmaxa 4D system. Cells were collected at two days post electroporationfor cDNA preparation and evaluated for transfection efficiency using aBeckman Coulter CytoFlex S cytometer. Transfection efficiency exceeded90%. Additional time points were collected at five- and eight-days postelectroporation for gDNA extractions. gDNA was prepared using theMachercy Nagel NucleoSpin Blood QuickPure kit.

Digital droplet PCR was utilized to determine the frequency of targetinsertions and deletions (indel %) using primers P1, F1, and R1 at theDMD 19-20 binding site; primers P3, F3 & R3 at the DMD 37-38 bindingsite; Primers P4, F4 and R4 at the 35-36 target site to generate anamplicon surrounding the binding site, primers P5, F5 & R5 at the DMD29-30 binding site, as well as primers P2, F2, R2 to generate areference amplicon. Amplifications were multiplexed in a 20 uL reactioncontaining 1×ddPCR Supermix for Probes (no dUTP, BioRad), 250 nM of eachprobe, 900 nM of each primer, 5 U of HindIII-HF, and about 50 ngcellular gDNA. Droplets were generated using a QX100 droplet generator(BioRad). Cycling conditions were as follows: 1 cycle of 95° C. (2° C./sramp) for 10 minutes, 44 cycles of 94° C. (1° C./s ramp) for 30 seconds,X° C. (1° C./s ramp) for 45 seconds (See below per “X” target siteannealing temperatures), 72° C. (0.2° C./s ramp) for 2 minutes, 1 cycleof 98° C. for 10 minutes, 4° C. hold. Droplets were analyzed using aQX200 droplet reader (BioRad) and QuantaSoft analysis software (BioRad)was used to acquire and analyze data. Indel frequencies were calculatedby dividing the number of positive copies for the binding site probe bythe number of positive copies for the reference probe and comparing lossof FAM+ copies in nuclease-treated cells to mock-transfected cells.

Cycling annealing temperatures: DMD 19-20: 57° C.; DMD 35-36 59° C.; DMD37-38 57° C.; and DMD 29-30 62° C.

TABLE 10 Primer and Probe Sets Sites Primer Seq Id No. Primer SequenceProbe 19-20 SEQ ID NO: 133 CGGGAGGTAATACATAATCC 19-20 F1 SEQ ID NO: 134GGGTGGGTTGCTTTACCTCTC 19-20 R1 SEQ ID NO: 135 TGGGCTACTGCAACTCTGTTP2 Reference SEQ ID NO: 136 AGGACAAAAGAGGACGGTCTGCCCTGG Reference F2SEQ ID NO: 137 TAAGACCCAGCTTCACGGAG Reference R2 SEQ ID NO: 138TATGATCGCCTGTTCCTCCA P3 37-38 SEQ ID NO: 139 CTGGCCGAAGTATAGGAA 37-38 F3SEQ ID NO: 140 CGCAACATGTGACATAAAGAG 37-38 R3 SEQ ID NO: 141TCTGGATATCCTCTTCTGGG P4 35-36 SEQ ID NO: 142 CCTACATGGTGTATCTGAC35-36 F4 SEQ ID NO: 143 GAACACCACCAGAAAAACAAG 35-36 R4 SEQ ID NO: 144CACTTCCTGTAAGACAACCAG P5 29-30 SEQ ID NO: 145 ATCCCTCATACCCAATC 29-30 F5SEQ ID NO: 146 AAAAACCACGGTGCTGTTGA 29-30 R5 SEQ ID NO: 147ATGGGGTCCGAGACTTTTCC

2. Results

Meganucleases targeting the DMD 19-20, DMD 29-30, DMD 35-36, and DMD37-38 recognition sequences were screened for their ability to generateindels in the MRC5 cell line. The DMD 19-20x.13 and DMD 19-20x.87meganucleases each returned a measurable amount of indel activity at thelow mRNA dose, with the DMD 19-20x.87 meganuclease showing more potencyin this single nuclease assay (FIG. 7A). The DMD 35-36x.63 and DMD35-36x.81 meganucleases each returned greater that 30% genomic editingin MRC5 cells at the low mRNA dose (FIG. 7B). The DMD 37-38x.15, DMD37-38x.66, and DMD 37-38x.79 meganucleases returned a >30% genomicediting at the low mRNA dose with no editing reduction over time (FIG.7C). The DMD 29-30x.18 meganuclease initially had high % indels, butthese were reduced significantly at day 8, while the DMD 29-30x.40meganuclease had a lower % of indels that were stable out to day 8 (FIG.7D).

3. Conclusions

These experiments demonstrated that engineered DMD meganucleases of theinvention were capable of generating indels at their recognitionsequences in cultured cells, with many capable of producing highfrequencies of indels that were stable over time, even when cells weretransfected with low concentration of mRNA.

Example 3 Characterization of Pairs of Meganucleases for Removing Exons45-55 of the Dystrophin Gene 1. Methods

These experiments evaluated pairs of engineered meganucleases targetingthe DMD 19-20, DMD 35-36, and DMD 37-38 recognition sequences for theirability to excise exons 45-55 of the dystrophin gene from the genome ofMRC5 cells. To accomplish this, MRC5 cells were transfected withseparate mRNAs encoding DMD 19-20 and DMD 35-36 meganucleases, orseparate mRNAs encoding DMD 19-20 and DMD 37-38 meganucleases. The pairsof DMD 19-20 and DMD 35-36 meganucleases tested included the following:DMD 19-20x.13 and DMD 35-36x.63; DMD 19-20x.87 and DMD 35-36x.81; DMD19-20x.13 and DMD 35-36x.81; DMD 19-20x.87 and DMD 35-36x.63. The pairsof DMD 19-20 and DMD 37-38 meganucleases tested included the following:DMD 19-20x.13 and DMD 37-38x.15; DMD 19-20x.87 and DMD 37-38x.15; DMD19-20x.13 and DMD 37-38x.66; DMD 19-20x.87 and DMD 37-38x.66; DMD19-20x.13 and DMD 37-38x.79; and DMD 19-20x.87 and DMD 37-38x.79.Additionally, these experiments also evaluated pairs of DMD 19-20 andDMD 29-30 meganucleases for their ability to excise only exon 45 of thedystrophin gene from the genome of MRC5 cells. The pairs of DMD 19-20and DMD 29-30 meganucleases tested included the following: DMD 19-20x.13and DMD 29-30x.18; DMD 19-20x.87 and DMD 29-30x.40; DMD 19-20x.13 andDMD 29-30x.40; and DMD 19-20x.87 and DMD 29-30x.18.

1e6 MRC5 cells were electroporated with 40 ng mRNA encoding each DMDmeganuclease or GFP using the Lonza Amaxa 4D system. Cells werecollected at two days post electroporation for gDNA preparation andevaluated for transfection efficiency using a Beckman Coulter CytoFlex Scytometer. Transfection efficiency exceeded 90%. gDNA was prepared usingthe Macherey Nagel NucleoSpin Blood QuickPure kit.

gDNA was PCR amplified with primer sets spanning the deletion sites toamplify any deletion event. The following PCR Primers sets in the tablebelow were used:

TABLE 11 Primer Sets Sites Primer Seq Id No. Primer Sequence19-20 to 35-36 SEQ ID NO: 148 GGGTGGGTTGCTTTACCTCTC SEQ ID NO: 149AGAGCATGCCATCTGAGTC 19-20 to 35-36 SEQ ID NO: 150 GTGAAGTAGCAAAGCACCTGSEQ ID NO: 151 AGAGCATGCCATCTGAGTC 19-20 to 29-30 SEQ ID NO: 152GGGTGGGTTGCTTTACCTCTC SEQ ID NO: 153 TTTGGTATGGGGTCCGAGAC 19-20 to 29-30SEQ ID NO: 154 GTGAAGTAGCAAAGCACCTG SEQ ID NO: 155 TTTGGTATGGGGTCCGAGAC19-20 to 37-38 SEQ ID NO: 156 GGGTGGGTTGCTTTACCTCTC SEQ ID NO: 157GATTCTCAGAAATGGAGTGACTG 19-20 to 37-38 SEQ ID NO: 158GTGAAGTAGCAAAGCACCTG SEQ ID NO: 159 GATTCTCAGAAATGGAGTGACTG

Q5 Taq Polymerase (New England Biolabs) was used in conjunction with theprimers above and dNTPs (New England Biolabs) to amplify gDNA. Thecycling conditions were as follows: 1 cycle of 98° C. for 4 minutes, 35cycles of 98° C. for 10 seconds, X° C. (see above TM for anneal) for 20seconds, 72° C. for 1.5 minutes, 1 cycle of 72° C. for 2 minutes, 4° C.hold. PCR products were run out on a 2% agarose gel and visualized by UVcamera. The remaining PCR products were used for sanger sequencing usingthe same primers utilized in the amplification step. DNA sequence wasanalyzed using the SnapGene Software and compared to the anticipatedtemplate file.

Digital droplet PCR was utilized to determine the frequency of largedeletions (indel %) utilizing primer pairs and probes that span thejunction of the 19-20 target site and the corresponding DMD nuclease foreither 29-30, 35-36, or 37-38. This assay measures the perfect ligationevent of the left half-site of 19-20 with the corresponding righthalf-site of either the 29-30, 35-36, or 37-38 nuclease site. With eachdual nuclease assay, the same reference amplicon assay from Example 2 asusing primers P2, F2, R2 was included allowing us to quantify the ratioof large deletion to a region unaffected by the nuclease activity. Asummary schematic can be found in FIG. 11 , which illustrates this assayfor the 19-20 to 37-38 dual nuclease delivery. The perfect ligation forDMD 19-20 and DMD 37-38 utilized primers P6, F6, and R6. The perfectligation for the DMD 19-20 and DMD 35-36 utilized primers P7, F7 and R7.The perfect ligation for DMD 19-20 to 29-30 utilized primers and probesP8, F8 and R8. Amplifications were multiplexed in a 20 uL reactioncontaining 1×ddPCR Supermix for Probes (no dUTP, BioRad), 250 nM of eachprobe, 900 nM of each primer, 5 U of HindIII-HF, and about 50 ngcellular gDNA. Droplets were generated using a QX100 droplet generator(BioRad). Cycling conditions were as follows: 1 cycle of 95° C. (2° C./sramp) for 10 minutes, 44 cycles of 94° C. (1° C./s ramp) for 30 seconds,X° C. (1° C./s ramp) for 45 seconds (see annealing temperature below pertarget site), 72° C. (0.2° C./s ramp) for 2 minutes, 1 cycle of 98° C.for 10 minutes, 4° C. hold. Droplets were analyzed using a QX200 dropletreader (BioRad) and QuantaSoft analysis software (BioRad) was used toacquire and analyze data. Indel frequencies were calculated by dividingthe number of positive copies for the binding site probe by the numberof positive copies for the reference probe and comparing loss of FAM+copies in nuclease-treated cells to mock-transfected cells.

Cycling annealing temperatures: DMD 19-20-DMD 37-38 at 53° C.,DMD19-20-DMD 37-38 at 59° C., and DMD 19-20-DMD 29-30 at 55° C.

TABLE 12 Primer and Probe Sets Sites Primer Seq ID NO. Primer SequenceP6 19/38 SEQ ID NO: 160 ATCAGAAGGATTATGTATAGGAATA 19-20 F6SEQ ID NO: 161 GGGTGGGTTGCTTTACCTCT 37-38 R6 SEQ ID NO: 162TCTGGATATCCTCTTCTGGG P7 19/36 SEQ ID NO: 163 GTGAAGTAGCAAAGCACCTG19-20 F7 SEQ ID NO: 164 GTGAAGTAGCAAAGCACCTG 35-36 R7 SEQ ID NO: 165AGTCACTTCCTAAGCTAAGACAACC P8 19/30 SEQ ID NO: 166CAGAAGGATTATGTATGAGGGATA 19-20 F8 SEQ ID NO: 167 GTGAAGTAGCAAAGCACCTG29-30 R8 SEQ ID NO: 168 ATGGGGTCCGAGACTTTTCC

2. Results

Correct sized PCR products were visualized for 19-20 to 35-36amplifications (FIG. 8A) with the corresponding sequence data showingthe perfect ligation between the 19-20/35-36 nuclease binding sites(FIG. 8B). The correct sized PCR products were also visualized for 19-20to 37-38 amplifications (FIG. 9A) with the corresponding sequence datashowing the perfect ligation between the 19-20/37-38 nuclease bindingsites (FIG. 9B). Furthermore, the correct sized PCR products werevisualized for 19-20 to 29-30 amplifications (FIG. 10 ).

The Digital droplet PCR (ddPCR) assays for all three deletion eventswere successful in measuring the perfect ligation between the 19-20recognition sequence and the corresponding sequence from either 29-30,35-36, or 37-38 with clean mock reactions. The perfect ligation for DMD19-20 to DMD 35-36 quantified deletion and ligation events ranged from 6to 12% (FIG. 12A). The perfect ligation for DMD 19-20 to DMD 37-38quantified deletion and ligation events ranged from 9 to 17.9% (FIG.12B) and the perfect ligation for DMD 19-20 to DMD 29-30 quantifieddeletion and ligation events ranged from 1 to 3% (FIG. 12C).

3. Conclusions

These results provide in vitro proof of concept for dual editing withremoval of the hot spot exons associated with multiple DMDdisease-associated mutations. These editing events were measured to beas high as 17.9%. However, the frequency of these edits, and the removalof either exon 45 alone or exons 45-55, are underestimated because thisassay only picks up the perfect ligation events, as opposed to ligationsthat result in minor indels at the target sites. Furthermore, it wasshown that this can be accomplished with multiple different engineeredmeganucleases and target sites with varying levels of success (FIG. 13). Unexpectedly, these data showed that the percentage of perfectligation was significantly higher for the removal of exons 45-55 andover 500,000 bp of genetic sequence with the DMD 19-20 and DMD 35-36/DMD37-38 meganuclease pairings, as compared to the removal of a single exon45 with the DMD 19-20 and DMD 29-30 meganuclease pairing (FIG. 12C andFIG. 13 ).

Example 4 Analysis of Perfect Ligation Events in the Dystrophin GeneFollowing Removal of Exons 45-55 in Primary Human Skeletal MuscleMyoblasts 1. Methods

In these experiments, the editing ability of meganucleases targeting theDMD 19-20 and DMD 37-38 recognition sequences was evaluated in humanskeletal muscle myoblasts, a human primary muscle cell. Human skeletalmuscle myoblasts were purchased from Lonza (HSMM CC-2580). Cells werethawed and seeded at 3500 cells/cm² in Skeletal muscle cell growthmedium-2 (SkGM-2) and maintained to a confluency not more than 70% untilelectroporation. Transfections were performed with 1e6 cells. Cells wereelectroporated with either 20, 40, 80 or 160 ng mRNA encoding each DMDmeganuclease in pairs (DMD 19-20L.249 and DMD 37-38L.166 pair) or GFPusing the Lonza Amaxa 4D system. After electroporation cells were seededinto growth media in individual wells of a 6 well plate. One day afterelectroporation, cells to be differentiated were changed to DMEMF12(Thermo) with 2% horse serum, while cells to be maintained asundifferentiated were maintained in growth media. Cells were harvested2- and 8-days post electroporation for DNA and protein extraction gDNAisolation and Digital droplet PCR was utilized to determine thefrequency of large deletions (indel %) for the DMD 19-20-DMD 37-38perfect ligation as specified in Example 3 above.

2. Results

A dose-dependent increase in the large deletion/perfect ligation wasseen with increasing amounts of DMD 19-20L.249 and DMD 37-38L.166engineered meganuclease mRNA in the HSMM cell lines (FIG. 14 ). Perfectligations following excision of exons 45-55 ranged from 14% to 27% forthe low to high dose mRNA.

3. Conclusions

Previous results of dual delivery large deletion were carried out in ahuman MRC5 cell line. By contrast, this experiment measured the abilityof engineered meganucleases of the invention to edit the genome atrecognition sites needed to excise the exons that contain the “Hot Spot”regions in a relevant muscle cell line. Increasing amounts of engineeredmeganuclease mRNA that targeted the DMD 19-20 and DMD 37-38 recognitionsequences were able excise a fragment greater than 500,000 bp andperfectly ligate the gene back together at frequencies up to 27%, asquantified by digital droplet PCR. These data support in vitro proof ofconcept of large-scale editing/deletion with a pair engineeredmeganucleases.

Example 5 Analysis of Large Deletion and Restoration of Dystrophin InVitro Following Increasing Amounts of Pairs of DMD Meganucleases in anImmortalized Cell Line Isolated from a DMD Patient 1. Methods

Pairs of DMD 19-20 and DMD 37-38 nucleases were further evaluated in aDMD patient cell line AB1098. The DMD patient myoblast cell line wasobtained from the Center for Research in Myology (Sorbonne University).This cell line was immortalized from the spinal muscle of a patient witha deletion of exons 48-50 and are dystrophin protein deficient due tothe deleted exons.

Cells were seeded at 3000 cells/cm² and grown in Promocell muscle growthmedia (Promocell). Transfections were performed with 1e6 cells in P5electroporation solution (Lonza) and transfected with the EY100 programusing the Lonza 4D-Nucleofector X unit. Nuclease mRNA dose was 10 ng, 20ng, 40 ng, 80 ng, or 160 ng for each of the DMD 19-20L.249 and DMD37-38L.166 meganucleases. After electroporation, cells were seeded intogrowth media in individual wells of a 6 well plate. One day afterelectroporation, cells to be differentiated were changed to DMEM(Thermo) 10 ug/ml Insulin, and 50 ug/ml gentamycin, while cells to bemaintained as undifferentiated were maintained in growth media. Cellswere harvested 2- and 8-days post electroporation for DNA and proteinextraction. gDNA isolation and Digital droplet PCR was utilized todetermine the frequency of large deletions (indel %) for the DMD19-20-DMD 37-38 perfect ligation as specified in Example 3 above. Forprotein extraction, cells were harvested from the plates with TrypLE,pelleted, then rinsed with PBS and lysed with 1×RIPA buffer withprotease inhibitors (Millipore). Protein concentration was determined byBCA assay (Thermo). For analysis by WES (Protein simple) lysates werenormalized to 250 ng/ul and run on the 66-440 kDa module using thestandard program. Primary antibody used for detection of dystrophin wasab154168 at 1:1000. Primary antibody vs Vinculin (Abcam) was used(1:200) as a loading control.

2. Results

A dose dependent increase in the large deletion/perfect ligation wasseen with increasing amounts of mRNA encoding the DMD 19-20L.249 and DMD37-38L.166 engineered meganucleases in this DMD patient cell line.Perfect ligations ranged from 4% to 29% (low to high dose mRNA) (FIG. 15).

Expression of the shortened modified dystrophin protein, lacking theamino acids encoded by exons 45-55, was measured by WES automatedwestern blot analysis. A dose dependent increase in the amount ofmodified dystrophin is viewed in the WES Chromatic readout, with thehighest dose 160 ng (line F), 80 ng (line E), 40 ng (line D), 20 ng(line C), ng (line B) and mock (line A) (FIG. 16A). The WES systemconverted the chromatic data generated into a more traditional WesternBlot figure and duplicated the read out. No dystrophin was detected inmock untreated AB1098 cells, with increasing intensity of bands fordystrophin across the dose range (FIG. 16B). Dystrophin restoration wasnormalized to a loading control Vinculin Protein and the amount ofprotein restoration was calculated relative to loading (FIG. 16C). Nodystrophin was measured in mock, while a seven-fold increase indystrophin was observed across the dose curve.

3. Conclusions

These experiments report the large deletion of exons 45 to 55 in a cellline isolated from a patient missing exons 48 to 50 in the dystrophingene. This cell line does not express detectable levels of dystrophinand is a good in vitro model for the DMD disease. The WES protein datain FIG. 16 shows restored expression of a shortened modified dystrophinprotein, with no protein expression in untreated mock cells to adetectable level across all engineered meganuclease dose ranges. This isfurther confirmation and in vitro proof of concept of using dualengineered meganucleases for the purpose of treating DMD patients byexcising exons 45-55 and converting the dystrophin gene to a Beckersdystrophin phenotype.

Example 6 Analysis of RNA Splice Message 1. Methods

This study characterized pairs of DMD 19-20 and DMD 37-38 meganucleasesin the patient cell line AB1098 looking at the RNA splice message postmeganuclease delivery.

Cells were cultured and electroporated as described in Example 5. Thedose of nuclease mRNA dose was 10 ng, 20 ng, 40 ng, 80 ng, or 160 ng foreach meganuclease (DMD 19-20L.249 and DMD 37-36L.166). Cells wereharvested on day 8 for RNA extraction using phenol chloroform and thePurelink RNA mini kit (Thermo). Post RNA isolation, cDNA synthesis wasperformed with the iSCRIPT cDNA synthesis kit (Bio-Rad) and ddPCR wasutilized to determine the frequency of the splicing of exon 44 to exon56. Dystrophin splice message of treated cells was normalized to areference message assay for the gene, Ankyrin Repeat Domain containingprotein 27 (ANKRD27) purchased from Thermo Fisher (assay#Hs01047624_g1).

CDNA Synthesis conditions: Primed for 5 minutes at 25° C., RT for 20minutes at 46° C. and inactivated for 1 minute at 95° C. ddPCR cycling95° C. for 10 minutes followed by cycles of 95° C. 45 seconds, 57° C. 45seconds, 72° C. for 1 minute and final inactivation of 98° C. for 10minutes.

TABLE 13 Primer and Probe Sets Sites Primer Seq Id No. Primer Sequence95 DMD Splice RNA For2 SEQ ID NO: 184 GCTGAACAGTTTCTCAGAAAGACA98 DMD RNA Splice Rev 2 SEQ ID NO: 185 GGCTGTTTTCATCCAGGTTGTG96 DMD 4456 RNA SPLICE PB SEQ ID NO: 186 TCTTAAGGACCTCCAAGG

2. Results

A dose-dependent increase in the exon 44 to exon 56 splice message wasseen with increasing amounts of DMD19-20L.249 and DMD 37-38L.166engineered meganuclease mRNA in the patient cell line. Splice message toreference gene message ratio ranged from 0.01% in the mock to 9.51% athigh dose mRNA (FIG. 17 ).

3. Conclusions

These experiments demonstrated the large deletion of exons 45 to 55 ofthe dystrophin RNA message in a cell line isolated from a DMD patient.This cell line does not express detectable levels of dystrophin and is agood in vitro model for DMD disease. The splice message data in FIG. 17shows the targeted deletion of exons 45 to 55. This is furtherconfirmation and in vitro proof of concept of using a dual engineeredmeganuclease strategy for the purpose of treating DMD patients byexcising exons 45-55, and restoring a “Beckers” dystrophin phenotype.

Example 7 Analysis of Perfect Ligation Events in the Dystrophin GeneFollowing Removal of Exons 45-55 in Primary Human Skeletal MuscleMyoblasts 1. Methods

These experiments evaluated the ability of meganucleases targeting theDMD 19-20 and DMD 35-36 recognition sequences to excise exons 45 to 55of the dystrophin gene in human skeletal muscle myoblasts, a humanprimary muscle cell, as described in Example 4. Cells were thawed andseeded at 3500 cells/cm² in Skeletal muscle cell growth medium-2(SkGM-2) and maintained to a confluency not more than 70% untilelectroporation. Transfections were performed with 1e6 cells. Cells wereelectroporated with either 40 ng mRNA encoding each DMD meganuclease inpairs (DMD 19-20x.13 and DMD 35-36x.63; DMD 19-20L.249 and DMD35-36L.195; DMD 19-20L.302 and DMD 35-36L.282; DMD 19-20L.329 and DMD35-36L.282; DMD 19-20L.302 and DMD 35-36L.349; or DMD 19-20L.329 and DMD35-36L.349) or GFP using the Lonza Amaxa 4D system. Afterelectroporation, cells were seeded into growth media in individual wellsof a 6 well plate. Cells were harvested 2 days post electroporation forDNA. gDNA isolation and digital droplet PCR was utilized to determinethe frequency of large deletions (indel %) for the DMD 19-20-DMD 35-36perfect ligation as specified in Example 3 above.

2. Results

As shown in FIG. 18 , each pair of engineered meganucleases demonstrateda deletion of exons 45-55 and perfect ligation of the dystrophin gene inthis primary myoblast cell line.

3. Conclusions

These results further demonstrate that pairs of engineered meganucleasesof the invention, which target the DMD 19-20 and DMD 35-36 recognitionsequences within the dystrophin gene, can excise exons 45-55 from thedystrophin gene and subsequently induce a perfect ligation of thedystrophin gene. These data are consistent with data provided in Example4 and further support in vitro proof of concept of large-scale editingand deletion with engineered meganucleases.

Example 8 Generation of Additional DMD 19-20 Meganucleases 1. Methods

Additional DMD 19-20 meganucleases were created after three rounds ofdevelopment to increase meganuclease potency and specificity.Meganuclease specificity was measured using the OligoCapture assay. Thisis a cell-based, in vitro assay that relies on the integration of asynthetic oligonucleotide (oligo) cassette at DSBs within the genome.Using the oligo as an anchor, genomic DNA to either side of theintegration site can be amplified, sequenced, and mapped (FIG. 19 ).This allows for a minimally biased assessment of potential off-targetediting sites of the nuclease. This technique was adapted from GuideSeq(Tsai et al. (2015) Nat. Biotech. 33:187-97) with specific modificationto increase sensitivity and accommodate the 3′ complementary overhangsinduced by the meganucleases. The OligoCapture analysis software issequence agnostic. That is, no a priori assumptions are made regardingwhich DNA sequences the nuclease is capable of cutting. In theOligoCapture assay, cells are transfected with nuclease mRNA anddouble-stranded DNA oligonucleotides. After 2 days, the cellular genomicDNA was isolated and sheared into smaller sizes. An oligonucleotideadapter was ligated to the sheared DNA and polymerase chain reaction wasused to amplify any DNA pieces that contain an adapter at one end andthe captured oligonucleotide at the other end. The amplified DNA waspurified, and sequencing libraries were prepared and sequenced. The datawere filtered and analyzed for valid sites that captured anoligonucleotide to identify potential off-target sites. The sequencereads were aligned to a reference genome, and grouped sequences withinthousand-base pair windows scanned for a potential meganuclease cleavagesite. HEK293 cells were transfected with mRNA for multiple DMD19-20nucleases at each round of optimization (rounds 1-3) gDNA was isolatedand processed as written above in the assay description.

2. Results

As shown in FIG. 20 , each off-target site generated by each DMD 19-20meganuclease in HEK293 cells is plotted based on the number of uniquesequence reads for a probe oligo being captured at that site with thedot cluster on the left representing low read counts and dots to theright representing high read counts. The specificity of the DMD 19-20meganucleases can be judged by how many intermediate sites are found inthe middle region of the graph and how low their read counts are. Fewerdots correlate to fewer detected potential off-target sites overall, anddots closer to the left correlate to lower read counts and lessconfidence that they are legitimate off-targets. The intended DMD targetsites should have the highest read counts, which is the case for bothnucleases selected for inclusion in DMD 19-20 meganuclease dots withinthe circles; also corresponding to fewer sequence mismatches compared tothe target site sequence shown by darker blue spots.

3. Conclusions

Meganuclease specificity for the 19-20 target site increased over threedevelopment rounds. These meganucleases had a specificity profile thatwarranted characterizing targeted off-targets.

Example 9 Generation of Additional DMD 35-36 Meganucleases 1. Methods

Additional DMD 35-36 meganucleases were created after three rounds ofdevelopment to increase meganuclease potency and specificity. Nucleasespecificity was measured using the OligoCapture assay as described inExample 8.

2. Results

As shown in FIG. 21 , each off-target site generated by each DMD 35-36meganuclease in HEK293 cells is plotted based on the number of uniquesequence reads for a probe oligo being captured at that site with thedot cluster on the left representing low read counts and dots to theright representing high read counts. The specificity of the DMD 35-36meganucleases can be judged by how many intermediate sites are found inthe middle region of the graph and how low their read counts arc. Fewerdots correlate to fewer detected potential off-target sites overall, anddots closer to the left correlate to lower read counts and lessconfidence that they are legitimate off-targets. The intended DMD targetsites should have the highest read counts, which is the case for bothnucleases selected for inclusion in DMD35-36 meganuclease dots withinthe circles also corresponding to fewer sequence mismatches compared tothe target site sequence shown by darker blue spots.

3. Conclusions

Meganuclease specificity for the 35-36 target site increased over 3development rounds. These generated meganucleases had a specificityprofile that warranted characterizing targeted off-targets.

Example 10 Editing of Dystrophin Gene in DMD Patient Cell Line UsingPairs of DMD Meganucleases 1. Methods

Pairs of improved DMD 19-20, DMD 35-36 and DMD 37-38 meganucleases werefurther evaluated in a DMD patient cell line AB1098. The DMD patientmyoblast cell line was obtained from the Center for Research in Myology(Sorbonne University). This cell line was immortalized from the spinalmuscle of a patient with a deletion of exons 48-50 and are dystrophinprotein deficient due to the deleted exons. Cells were transduced withpairs of the engineered meganucleases targeting the DMD 19-20 and DMD35-36 or the DMD 19-20 and DMD 37-38 recognition sequences as shown inTable 14 below.

TABLE 14 Pairs of Engineered Meganucleases EP 19-20/35-36 Pair 1 19-20L.374/35-36 L.376 2 19-20 L.374/35-36 L.457 3 19-20 L.374/35-36 L.469 419-20 L.375/35-36 L.376 5 19-20 L.375/35-36 L.457 6 19-20 L.375/35-36L.469 7 19-20 L.431/35-36 L.376 8 19-20 L.431/35-36 L.457 9 19-20L.431/35-36 L.469 10 19-20 L.458/35-36 L.376 11 19-20 L.458/35-36 L.45712 19-20 L.458/35-36 L.469 13 Mock 14 19-20 L.374/37-38 L.478 15 19-20L.374/37-38 L.512 16 19-20 L.374/37-38 L.528 17 19-20 L.375/37-38 L.52818 19-20 L.375/37-38 L.528 19 19-20 L.375/37-38 L.528 20 19-20L.431/37-38 L.528 21 19-20 L.431/37-38 L.528 22 19-20 L.431/37-38 L.528

Cells were seeded at 3000 cells/cm² and grown in Promocell muscle growthmedia (Promocell). Transfections were performed with 1e6 cells in P5electroporation solution (Lonza) and transfected with the EY100 programusing the Lonza 4D-Nucleofector X unit. Meganuclease mRNA dose was 40 ngfor each of the DMD 19-20, DMD 35-36, and DMD 37-38 mcganuclcases. Afterelectroporation, cells were seeded into growth media in individual wellsof a 6 well plate. One day after electroporation, cells to bedifferentiated were changed to DMEM (Thermo) 10 ug/ml insulin, and 50ug/ml gentamycin, while cells to be maintained as undifferentiated weremaintained in growth media. Cells were harvested 2- and 8-days postelectroporation for DNA and protein extraction. gDNA was isolated andDigital droplet PCR was utilized to determine the frequency of largedeletions (indel %) for the DMD 19-20-DMD 35-36 and DMD 19-20-DMD 37-38meganucleases. The reagents, cycling conditions and reference assay wereconducted as described in example 2 switching out the PCR primers andprobes to measure total ligation. This ddPCR assay used a forward primer5′ of the 19-20 binding site and a reverse primer 3′ to the 35-36 siteor to the 37-38 site and a probe specific to the sequence 51 base pairs5′ to the ligated 19-20/35-36 site or the 19-20/37-38 site (see, Table15 below). With each dual meganuclease assay, the same referenceamplicon assay from Example 2 was included allowing quantification ofthe ratio of large deletion to a region unaffected by the pair ofmeganucleases (see, Table 15 below).

For protein extraction, cells were harvested from the plates withTrypLE, pelleted, then rinsed with PBS and lysed with 1×RIPA buffer withprotease inhibitors (Millipore). Protein concentration was determined byBCA assay (Thermo). For analysis by WES (Protein simple) lysates werenormalized to 250 ng/ul and run on the 66-440 kDa module using thestandard program. Primary antibody used for detection of dystrophin was1:50 mandyS10. Primary antibody vs Vinculin (Abeam) was used (1:100) asa loading control.

TABLE 15 Primers used in ddPCR assay for total ligation determinationPrimer Name Primer Sequence SEQ ID NO. 143 DMDligfor4GGGTGGGTTGCTTTACCTCTCTAG SEQ ID NO: 187 145 DMD1936REVTCACATCATGAGATTTAGTCACTTCC SEQ ID NO: 188 141 DMD 38 revers2GCTATCTGGATATCCTCTTCTGGG SEQ ID NO: 193 134 DMDimpLIG probeTTGCTACTTCACAGTAACCACATGG SEQ ID NO: 189 P2 ReferenceAGGACAAAAGAGGACGGTCTGCCCTGG SEQ ID NO: 136 Reference F2TAAGACCCAGCTTCACGGAG SEQ ID NO: 137 Reference R2 TATGATCGCCTGTTCCTCCASEQ ID NO: 138

2. Results

Varying levels of large deletion/total ligation were seen with differentpairings of the DMD 19-20 and DMD 35-36 or DMD 19-20 and DMD 37-38engineered meganucleases in this DMD patient cell line (FIG. 22 and FIG.24A). Total ligations ranged from 8% to 22.7% with the DMD 19-20 and DMD35-36 pairing (FIG. 22 ) and from a little over 1% to 12% with the DMD19-20 and DMD 37-38 pairing (FIG. 24A). Expression of the shortenedmodified dystrophin protein, lacking the amino acids encoded by exons45-55, was measured by WES. The WES system converted the chromatic datagenerated into a more traditional Western Blot figure and duplicated theread out. No dystrophin was detected in Mock untreated AB1098 cells.Shortened modified dystrophin was detected for each of the DMD 19-20 andDMD 35-36 engineered meganuclease pairs (FIG. 23A). Shortened modifieddystrophin restoration was normalized to a loading control VinculinProtein and the amount of protein restoration was calculated relative toloading (FIG. 23B). Similarly, shortened modified dystrophin wasdetected for cells treated with the pairing of DMD 19-20 and DMD 37-38engineered meganucleases compared to the relative expression level of anequal load (500 ng) of lysate from hDMD mouse quadriceps muscle based ona standard curve generated from that tissue (FIG. 24B). No dystrophinwas measured in mock, whereas shortened modified dystrophin was detectedin each of the meganuclease pairs.

3. Conclusions

These experiments report the large deletion of exons 45 to 55 in a cellline isolated from a patient missing exons 48 to 50 in the dystrophingene. This cell line does not express detectable levels of dystrophinand is a good in vitro model for the DMD disease. The WES protein data(FIG. 23A) and quantification in FIGS. 23B and 24B show restoredexpression of a shortened modified dystrophin protein with no proteinexpression in untreated mock cells to a detectable level across allengineered meganuclease dose ranges. This protein quantification isfurther confirmation and an in vitro proof of concept of using dualengineered meganucleases for the purpose of treating DMD patients byexcising exons 45-55 and converting the dystrophin gene to a Beckersdystrophin phenotype.

Example 11 Editing of Dystrophin Gene In Vivo in hDMD Mouse Study(TD066) 1. Methods

An in vivo study in hDMD mice was conducted to investigate in vivoediting and human dystrophin protein restoration induced by delivery ofthe pair of DMD 19-20x.13 and DMD 37-38x.15 meganucleases. Mice wereinjected by retro orbital systemic injection with three differentconstructs encapsulated with AAV9 (1×10¹⁴ VG/kg). The first AAV wascomprised of a viral genome that includes, from 5′ to 3′, the A17-120enhancer, the muscle-specific promoter MHCK7, a SV40 intron sequence,the coding sequence for the DMD 19-20x.13 nuclease, a furin GSG P2Acleavage sequence, a coding sequences for the DMD37-38x.15 nuclease, aWPRE element, and an SV40 poly adenylation signal. Additionally, twoAAV9 Constructs were investigated using a two-cassette approach whereeach nuclease is driven off a separate muscle specific promoter. Eachviral genome comprised of either a CK8 or MHCK7 muscle specific promoterat the 5′ end just after the ITR, then a HBA2 5′UTR, the coding sequencefor the DMD 19-20x.13 nuclease, a WPRE element, an SV40 poly adenylationsignal, then the muscle specific promoter SPc5-12, an XBG 5′ UTRsequence, the coding sequence for the DMD 37-38x.15 nuclease, an XBG 3′UTR sequence and a BgH poly adenylation signal. At 14 dayspost-injection, mice were sacrificed and tissue sections from skeletalmuscle (quadricep), heart, diaphragm, soleus, and liver were collectedfor molecular and histological analysis. The ddPCR assay of example 10used a forward primer 5′ of the 19-20 binding site, a reverse primer 3′to the 35-36 site and probe specific to sequence 51 base pairs 5′ to theligated 19-20/35-36 site. The perfect ligation assay in this exampleused a probe sequence specific to a perfect ligation of the 19-20 and35-36 binding sites with a comparable forward and reverse primer pair (aforward primer 5′ of the 19-20 binding site, a reverse primer 3′ to the35-36 site; Table 16).

TABLE 16 Primers used in ddPCR assay for perfect ligation determinationPrimer Name Primer Sequence SEQ ID NO. 49 DMD 19-20 F1GGGTGGGTTGCTTTACCTCT SEQ ID NO: 190 40-DMD 37-38 R TCTGGATATCCTCTTCTGGGSEQ ID NO: 191 89 DMD 1938 Probe ATCAGAAGGATTATGTATAGGAATASEQ ID NO: 192 P2 Reference AGGACAAAAGAGGACGGTCTGCCCTGG SEQ ID NO: 136Reference F2 TAAGACCCAGCTTCACGGAG SEQ ID NO: 137 Reference R2TATGATCGCCTGTTCCTCCA SEQ ID NO: 138

2. Results

The single promoter MHCK7 P2A AAV had successful perfect ligation eventsacross all tissues; averaging 2.8% in the quadricep, 5.3% in the heart,0.85% in the diaphragm, 3.9% in the soleus & 2.4% in the liver. TheCK8/SPc5-12 double promoter AAV had successful perfect ligation in alltissues except the liver, averaging 5.5% in the quadricep, 5.9% in theheart, 2% in the diaphragm, 1.45% in the soleus & 0.3% in the liver. TheMHCK7/SPC512 double promoter AAV had minimal perfect ligation eventsaveraging 0.2% in the quadricep, 1.7% in the heart, 0.04% in thediaphragm, 1.45% in the soleus & 0.08% in the liver (FIG. 25A-25E).

3. Conclusions

Here we report in vivo excision of the hot spot region (exons 45-55) ina humanized DMD mouse model. The double promoter CK8+SPc5-12 combinationappears to have low off-organ editing in the liver & higher editing inquad, diaphragm, and heart.

Example 12 Editing of Dystrophin Gene In Vivo in hDMD Mouse Study(TD069) 1. Methods

An in vivo study in hDMD mice was conducted to investigate in vivoediting and human modified dystrophin protein restoration induced bydelivery of the pair of DMD 19-20L.329 and DMD 37-38L.219 meganucleases.Mice were injected by retro orbital systemic injection with fourdifferent constructs encapsulated with AAV9 (1×10¹⁴ VG/kg). The firstset of AAVs varied the 3′ downstream nuclease but kept all elementsequal between the two. These were comprised of viral genomes thatincludes, from 5′ to 3′, the A 17-120 enhancer, the muscle-specificpromoter CK8, an SV40 intron sequence, the coding sequence for the DMD19-20L.329 nuclease, a furin GSG P2A cleavage sequence, a codingsequence for the DMD35-36L.349 or DMD37-38L.219 nucleases, a WPREelement, and an SV40 poly adenylation signal. The next set of AAVsvaried the 3′ downstream nuclease but kept all elements equal betweenthe two. There were comprised of viral genomes that includes, from 5′ to3′, the A17-120 enhancer, the muscle-specific promoter CK8, the codingsequence for the DMD 19-20L.329 nuclease, a furin GSG P2A cleavagesequence, a coding sequence for the DMD35-36L.349 or DMD37-38L.219nucleases, a WPRE element, and an SV40 poly adenylation signal. At 14days post-injection, mice were sacrificed and tissue sections fromskeletal muscle (quadricep), heart, diaphragm, soleus, and liver werecollected for molecular and histological analysis. ddPCR was conductedas described in Example 10.

2. Results

The AAV containing A17-120 CK8 SV40 intron DMD19-20L.329 P2ADMD35-36L.349 had successful total ligation events across all tissues;averaging 3.3% in the quadricep, 5.7% in the heart, 2.6% in thediaphragm, 0.6% in the soleus & 4.1% in the liver. The AAV containingA17-120 CK8 SV40 intron DMD19-20L.329 P2A DMD37-38L.219 had minimaltotal ligation events across all tissues; averaging 0.7% in thequadricep, 1.6% in the heart, 1.4% in the diaphragm, 0.3% in the soleus& 1.3% in the liver. The AAV containing A17-120 CK8 DMD19-20L.329 P2ADMD35-36L.349 had successful total ligation events across all tissues;averaging 5.6% in the quadricep, 3.6% in the heart, 3.6% in thediaphragm, 4.3% in the soleus & 2.2% in the liver. The AAV containingA17-120 CK8 DMD19-20L.329 P2A DMD37-38L.219 had minimal total ligationevents across all tissues; averaging 2.1% in the quadricep, 3.4% in theheart, 1.2% in the diaphragm, 0.35% in the soleus & 0.93% in the liver(FIG. 26A-26E).

3. Conclusions

Here we report excision of exons 45-55 with the DMD 19-20 & DMD 35-36meganucleases used in Example 11 as well as with the paring the DMD19-20 nuclease with a different downstream nuclease at the DMD 37-38target site. In this experiment, the paired DMD 19-20 meganuclease andDMD 35-36 meganuclease had higher levels of editing than the DMD19-20and DMD 37-38 paired meganucleases. The addition of the SV40 introndownstream of the promoter increased editing minimally in target tissuesand increased editing in the liver.

Example 13 Editing of Dystrophin Gene In Vivo in hDMDdel52/C57 MouseStudy (TD075) 1. Methods

An in vivo study in hDMDdel52/mdx (hDMD mouse) mice was conducted toinvestigate in vivo editing and shortened modified human dystrophinprotein restoration induced by delivery of the pair of DMD 19-20x.13 andDMD 37-38x.15 meganucleases. Mice were injected by retro orbitalsystemic injection with one construct encapsulated with AAV9 at twodoses, 1×10¹⁴ VG/kg or 2×10¹⁴ VG/kg. The AAV was comprised of a viralgenome that includes, from 5′ to 3′, the muscle-specific promoter CK8,an SV40 intron sequence, the coding sequence for the DMD 19-20x.13meganuclease, a furin GSG P2A cleavage sequence, a coding sequence forthe DMD 37-38x.15 meganuclease, a WPRE element, and an SV40 polyadenylation signal. At 14 days post-injection, mice were sacrificed andtissue sections from skeletal muscle (quadricep), heart, diaphragm, TA,and liver were collected for molecular, protein, and histologicalanalysis. ddPCR was conducted as described in Example 10. Dystrophinprotein expression was assessed in the quadriceps, heart, and diaphragmof the nuclease-treated animals using the Wes™ system (ProteinSimple) asdescribed in Example 10. Dystrophin expression was normalized to thehouse keeping protein vinculin and measured against a standard curve ofdystrophin protein isolated from a hDMD mouse that expresses full-lengthdystrophin. Tissue sections from the nuclease-treated mice were alsosubjected to IHC analyses to visualize dystrophin and meganucleaseprotein expression.

2. Results

The AAV at the dose of 1×10¹⁴ VG/kg (1×10¹² total AAV) containing CK8,SV40 intron, and DMD19-20x.13 P2A DMD37-38x.15 had successful totalligation events across all tissues; averaging 4.3% in the quadricep,3.6% in the heart, 1.9% in the diaphragm, 2.5% in the TA, and 0.6% inthe liver. The AAV at the dose of 2×10¹⁴ VG/kg (4×10¹² total AAV)containing CK8, SV40 intron, and DMD19-20x.13 P2A DMD37-38x.15 hadsuccessful total ligation events across all tissues; averaging 3.4% inthe quadricep, 2.7% in the heart, 3.9% in the diaphragm, 2.8% in the TA& 1.9% in the liver (FIGS. 27A-27E). Dystrophin restoration wasquantified by comparing dystrophin protein from the correspondingtissues in hDMD mice from a protein standard by a standard curve fromWES protein analysis. As shown in FIGS. 28A-28C lanes 1-6 represent thestandard curve; lanes 7-8 represent muscle tissue from the hDMD mousetreated with 1×10¹⁴ VG/kg of the DMD19-20x.13 and DMD37-38x.15meganucleases; lanes 9-10 represent muscle treated with 2×10¹⁴ VG/kg ofthe DMD19-20x.13 and DMD37-38x.15 meganucleases, and lanes 11-12represent mice treated with PBS. Based on the standard curve, treatedmice were found to average 6.5% dystrophin in the heart, 1.5% dystrophin(low dose) or 3% dystrophin (high dose) in the diaphragm, and 4.5%dystrophin in the quadricep (FIG. 28A-28C).

3. Conclusions

Here we report in vivo excision of the hot spot region (exons 45-55) ina humanized DMD mouse model with deletions in the human dystrophin gene.This genotype is an example of what could be found in a disease model orpatient. No significant difference in editing was seen with the two doselevels. This mouse model does not make human dystrophin as seen in lanes11 and 12 of FIGS. 28A-28C. Here, we report proof of concept ofshortened modified human dystrophin restoration in an in vivo model thatdoes not make human dystrophin due to mutations.

Example 14 Editing of Dystrophin Gene In Vivo in hDMDdel52/MDX MouseStudy (TD073) 1. Methods

An in vivo study in hDMDdel52/mdx (hDMD) mice was conducted toinvestigate in vivo editing and shortened modified human dystrophinprotein restoration induced by delivery of the DMD 19-20L.329 and DMD35-36L.349 pair of meganucleases. Mice were injected by retro orbitalsystemic injection with three different constructs encapsulated withAAV9 (1×10¹⁴VG/kg). The first AAV was comprised of a viral genome thatincludes, from 5′ to 3′, the A17-120 enhancer, the muscle-specificpromoter CK8, the coding sequence for the DMD 19-20L.329 meganuclease, afurin GSG P2A cleavage sequence, a coding sequence for the DMD35-36L.349meganuclease, a WPRE element, and an SV40 poly adenylation signal. Thesecond AAV was comprised of a viral genome that includes, from 5′ to 3′,the A17-120 enhancer, the muscle-specific promoter MHCK7, the codingsequence for the DMD 19-20L.329 nuclease, a furin GSG P2A cleavagesequence, a coding sequence for the DMD35-36L.349 nuclease, a WPREelement, and an SV40 poly adenylation signal. The third AAV wascomprised of a viral genome that includes, from 5′ to 3′, themuscle-specific promoter CK8, the coding sequence for the DMD 19-20L.329nuclease, a WPRE element, an SV40 poly adenylation signal, the musclespecific promoter SPc5-12, a coding sequence for the DMD35-36L.349nuclease, a WPRE element, and a BgH poly adenylation signal. At 14 dayspost-injection, mice were sacrificed and tissue sections from skeletalmuscle (quadricep), heart, diaphragm, and liver were collected formolecular, protein, and histological analysis. The ddPCR assay wasconducted as described in Example 10. Dystrophin protein expression wasassessed in the quadriceps, heart, and diaphragm of the nuclease-treatedanimals using the Wes™ system (ProteinSimple) as described in Example10. Dystrophin expression was normalized to the house keeping proteinvinculin and measured against a standard curve of dystrophin proteinisolated from a hDMD mouse that expresses full-length dystrophin.

Quadricep tissue sections from the nuclease-treated mice were alsosubjected to IHC analyses to visualize dystrophin and meganucleaseprotein expression. Briefly, quadricep tissues were dewaxed and treatedwith HEIR (Heat-Induced Epitope Retrieval) with ER1 for 40 min on theBOND RX. Slides were blocked in 10% NGS PBST (PBS with 0.1% Tween20)with MoM (Mouse on Mouse Blocking reagent, VECTOR) blocking reagent for1 h at room temp then incubated with Rabbit monoclonal anti-meganucleaseantibody (PBI, Rab54) at a dilution of 1:1500 and Mouse Monoclonalanti-Pax7 antibody (DSHB, Supernatant, 1:5) in PBST with 2% NGS at 4 Covernight in a humid chamber. The next day samples were incubated withSecondary antibodies (Goat-anti-mouse IgG1 Alexa647 (Invitrogen)),goat-anti-rabbit Alexa555 (Invitrogen)., 1:500) for 1 hr at roomtemperature followed by DAPI nuclear counterstained for 5 min. ExcessBOND RX wash buffer was removed, coverslips were mounted usingVectaShield Vibrance Antifade Mounting Medium. Imaging was performed onZeiss Apotome 2.0

BaseScope analysis for co-expression of Pax7 RNA and the dystrophinspliced message of exons 44 and 56 was performed on quadriceps tissuesections. This technique utilizes a detectable RNA probe that isapproximately 50 base pairs spanning the exon 44 and exon 56 junction(obtained from Advanced Cell Diagnostics (ACD), Newark, CA), which iscreated after genome repair via direct re-ligation (FIG. 34 ). Slideswere dewaxed and incubated with RNAscope hydrogen peroxide solution for10 min at room temperature followed by target retrieval in RNAscope 1×Target Retrieval Reagent in a steamer for 15 min at 95° C. and ProteaseTV treatment for 30 minutes at room temperature. The probes for thedystrophin exon44-56 junction and Pax7 were added and incubated in aHybEZ oven for 2 hours at 40° C. then stored overnight in 5×SSC. Signalwas enhanced using the BaseScope Duplex Assay (ACD) per themanufacturer's protocol followed by hematoxylin staining and imaging ofslides on a Leica GT450 digital scanner.

2. Results

Total ligation averaged 15% in the quadricep and ˜4% in the heart anddiaphragm, relative to the reference site (FIG. 29 ). Shortened modifieddystrophin restoration was quantified by comparing dystrophin proteinfrom the corresponding tissues in hDMD mice using a standard curve (FIG.30A-30C). Treated mice were found to average 8.2% in the quadricep, 4.2%in the heart, and 2.8% in the diaphragm (FIG. 31 ). In hDMD mice treatedwith PBS, no human dystrophin or meganuclease expression was detected inquadricep tissue. Notably, in hDMD mice exposed to DMD19-20L.329 andDMD35-36L.349, the expression of shortened modified human dystrophin wasrestored, and meganuclease protein staining was present in adjacenttissue sections (FIG. 32 ). Immunofluorescence staining of quadriceptissue sections show minimal background staining for nuclease in PBStreated animals and clear Pax7 staining of satellite cells (white arrowheads) (FIG. 33A). In nuclease treated animals, there is co-staining ofa population of Pax7 positive cells indicating expression of thenuclease in satellite cells (FIG. 33B). BaseScope analysis showedco-expression of Pax7 and the spliced RNA product (exon 44-56) intreated animals (FIG. 35B) compared with Pax7 alone in PBS animals (FIG.35A).

3. Conclusions

This study demonstrated in vivo proof of concept for excision of exons45 to 55 & dystrophin protein restoration in a DMD disease model.

Example 15 Restoration of Dystrophin Protein Expression in DMD PatientCell Lines 1. Methods

Previously we reported dystrophin protein restoration in a DMD patientcell line, AB1098, missing exons 48-50 as well as dystrophin expression.Here we report similar results with improved DMD 19-20L.329 and DMD35-36L.349 meganucleases in an additional KM1328 DMD patient cell line.The DMD patient myoblast cell line was obtained from the Center forResearch in Myology (Sorbonne University). This cell line wasimmortalized from the spinal muscle of a patient with a deletion of exon52, which causes it to be dystrophin protein deficient due to thedeleted exons. Cells were electroporated, cultivated and harvested aswritten in Example 10 previously. The meganuclease mRNA dose was 20 ng,80 ng and 160 ng of DMD 19-20L.329 and DMD 35-36L.349 meganucleases. Forcomparison, AB1098 patient cells were transfected with 80 ng of DMD19-20L.329 and DMD 35-36L.349 meganucleases. Protein was extracted,quantified and analyzed using the WES system as written in Example 10previously. Primary antibody used for detection of dystrophin was 1:50mandyS106. Primary antibody vs vinculin (Abcam) was used (1:100) as aloading control.

2. Results

Expression of the shortened modified dystrophin protein, lacking theamino acids encoded by exons 45-55, was measured by WES automatedwestern blot analysis. A dose-dependent increase in the amount ofmodified dystrophin was seen. The shortened modified dystrophin proteinband can be visualized in lanes 3-5 of FIG. 36 and lane 3 of FIG. 37 .The protein band for full length dystrophin for comparison can bevisualized in lane 6 of FIG. 36 and lane 1 of FIG. 37 . No dystrophinwas detected in mock untreated KM1328 cells, with increasing intensityof bands for dystrophin across the dose range (FIG. 36 ).

A dose-dependent increase in the large deletion/perfect ligation wasseen with increasing amounts of mRNA encoding the DMD 19-20L.329 and DMD35-36L.349 engineered meganucleases in KM1328 cells. This ligationresulted in increased detection of shortened modified dystrophin atincreasing doses of the pair of meganucleases (FIG. 36 ). Similarly,expression of the shortened modified dystrophin was observed with the 80ng dose of the pair of meganucleases in AB1098 cells (FIG. 37 ).

3. Conclusions

These experiments report the large deletion of exons 45 to 55 in a cellline isolated from multiple DMD cell lines with deletions in thedystrophin gene. These cell lines do not express detectable levels ofdystrophin and are a good in vitro model for the DMD disease. The WESprotein data in FIG. 36 and FIG. 37 show restored expression of ashortened modified dystrophin protein with no protein expression inuntreated mock cells to a detectable level across all engineeredmeganuclease dose ranges. This is further confirmation and in vitroproof of concept of using dual engineered meganucleases for the purposeof treating DMD patients by excising exons 45-55 and converting thedystrophin gene to a Beckers dystrophin phenotype.

Example 16 Editing of Dystrophin Gene In Vivo in PAX 7 Expressing MuscleCells in a hDMDdel52/MDX Mouse Study Using AAVrh74 1. Methods

This in vivo study was an evaluation of an AAV comprised of a viralgenome that includes, from 5′ to 3′, the A17-120 enhancer, themuscle-specific promoter MHCK7, the coding sequence for the DMD19-20L.329 nuclease, a furin GSG P2A cleavage sequence, a codingsequence for the DMD35-36L.349 nuclease, a WPRE element, and an SV40poly adenylation signal. The AAVrh74 containing the DMD 19-20L.329 andDMDL.349 meganucleases was compared to PBS delivered by retro orbitalsystemic injection with 1×10¹⁴, 3×10¹³, 1×10¹³ or 5×10¹² VG/kg inhDMDdel52/mdx (hDMD) mice. At 28 days post-injection, mice weresacrificed and tissue sections from skeletal muscle (quadricep), heart,diaphragm, and liver were collected for molecular, protein, andhistological analysis. Digital droplet PCR was conducted as described inExample 10. Dystrophin protein expression was assessed in thequadriceps, heart, and diaphragm of the nuclease-treated hDMDdel52/mdxanimals using the Wes™ system (ProteinSimple) as described in Example10. Dystrophin expression was normalized to the house keeping proteinvinculin and measured against a standard curve of dystrophin proteinisolated from a hDMD mouse that expresses full-length dystrophin. Tissuesections from the nuclease-treated mice were also subjected tofluorescent immunohistochemistry analyses to visualize dystrophin andmeganuclease protein expression as outlined in Example 14.

2. Results

Immunofluorescence staining of quadriceps tissue sections shows minimalbackground staining for nuclease in PBS treated animals and clear Pax7staining of satellite cells (white arrow heads) (FIG. 38A). In nucleasetreated animals, there is co-staining of a population of Pax7 positivecells indicating expression of the nuclease in satellite cells (FIG.38B). This study revealed expression of nuclease in Pax7 positive cellsacross all doses tested: 1×10¹⁴ VG/kg (FIG. 38B), 3×10³ VG/kg (FIG.38C), and 1×10³ VG/kg (FIG. 38D).

3. Conclusions

This study provides further proof of concept for editing the satellitecell population in the quadriceps muscle using an AAVrh74 encapsidatingthe dual meganucleases. The study showed coexpression of Pax7, a knownsatellite marker with expression of the engineered meganucleaseindicating that the meganuclease is expressed in this target populationand could therefore potentially edit these cells as previously shown inExample 14 with the same construct using the AAV9 capsid.

1. A polynucleotide comprising a nucleic acid sequence encoding anengineered meganuclease that binds and cleaves a nucleic acid at a sitethat comprises a recognition sequence consisting of the nucleic acidsequence of SEQ ID NO: 10 in a dystrophin gene, wherein said engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO:
 51. 2. Thepolynucleotide of claim 1, wherein said polynucleotide comprises apromoter operably linked to said nucleic acid sequence encoding saidengineered meganuclease.
 3. The polynucleotide of claim 2, wherein saidpromoter is a muscle-specific promoter.
 4. The polynucleotide of claim1, wherein said polynucleotide is an mRNA.
 5. A recombinant DNAconstruct comprising a polynucleotide comprising a nucleic acid sequenceencoding an engineered meganuclease that binds and cleaves a nucleicacid at a site that comprises a recognition sequence consisting of thenucleic acid sequence of SEQ ID NO: 10 in a dystrophin gene, whereinsaid engineered meganuclease comprises the amino acid sequence of SEQ IDNO:
 51. 6. The recombinant DNA construct of claim 5, wherein saidrecombinant DNA construct encodes a recombinant virus comprising saidpolynucleotide.
 7. The recombinant DNA construct of claim 6, whereinsaid recombinant virus is a recombinant adeno-associated virus (AAV). 8.The recombinant DNA construct of claim 7, wherein said recombinant AAVhas a capsid comprising the amino acid sequence of SEQ ID NO: 182 or acapsid comprising the amino acid sequence of SEQ ID NO:
 183. 9. Therecombinant DNA construct of claim 5, wherein said polynucleotidecomprises a promoter operably linked to said nucleic acid sequenceencoding said engineered meganuclease.
 10. The recombinant DNA constructof claim 9, wherein said promoter is a muscle-specific promoter.
 11. Therecombinant DNA construct of claim 6, wherein said polynucleotidecomprises a promoter operably linked to said nucleic acid sequenceencoding said engineered meganuclease.
 12. The recombinant DNA constructof claim 11, wherein said promoter is a muscle-specific promoter. 13.The recombinant DNA construct of claim 7, wherein said polynucleotidecomprises a promoter operably linked to said nucleic acid sequenceencoding said engineered meganuclease.
 14. The recombinant DNA constructof claim 13, wherein said promoter is a muscle-specific promoter. 15.The recombinant DNA construct of claim 8, wherein said polynucleotidecomprises a promoter operably linked to said nucleic acid sequenceencoding said engineered meganuclease.
 16. The recombinant DNA constructof claim 15, wherein said promoter is a muscle-specific promoter.
 17. Arecombinant virus comprising a polynucleotide comprising a nucleic acidsequence encoding an engineered meganuclease that binds and cleaves anucleic acid at a site that comprises a recognition sequence consistingof the nucleic acid sequence of SEQ ID NO: 10 in a dystrophin gene,wherein said engineered meganuclease comprises the amino acid sequenceof SEQ ID NO:
 51. 18. The recombinant virus of claim 17, wherein saidrecombinant virus is a recombinant AAV.
 19. The recombinant virus ofclaim 18, wherein said recombinant AAV has a capsid comprising the aminoacid sequence of SEQ ID NO: 182 or a capsid comprising the amino acidsequence of SEQ ID NO:
 183. 20. The recombinant virus of claim 17,wherein said polynucleotide comprises a promoter operably linked to saidnucleic acid sequence encoding said engineered meganuclease.
 21. Therecombinant virus of claim 20, wherein said promoter is amuscle-specific promoter.
 22. The recombinant virus of claim 18, whereinsaid polynucleotide comprises a promoter operably linked to said nucleicacid sequence encoding said engineered meganuclease.
 23. The recombinantvirus of claim 22, wherein said promoter is a muscle-specific promoter.24. The recombinant virus of claim 19, wherein said polynucleotidecomprises a promoter operably linked to said nucleic acid sequenceencoding said engineered meganuclease.
 25. The recombinant virus ofclaim 24, wherein said promoter is a muscle-specific promoter.